KR20060112186A - Direct detection method for products of cellular metabolism using tof-sims - Google Patents

Abstract

A rapid and efficient method for novel biological substance screening by surface analysis has been developed using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS). This method relies on the surface screening of an array of micro-organisms grown on porous membranes, which had previously been in contact with a solid growth medium. ToF-SIMS analysis differentiates among organisms producing different substances, either directly as molecular product, or indirectly through the use of multivariate statistical data reduction techniques. This method has many advantages over traditional microbial screening methods, which require sample preparation and time for assay development.

Description

Direct Detection Method for Products of Cellular Metabolism Using ToF-SIMS

This patent application claims the priority of US patent application USSN 60/502151, filed September 11, 2003.

The present invention relates to the field of microorganisms. More specifically, the present invention relates to a method of detecting cell products by time-of-flight mass spectrometry.

Many different techniques are currently available for analyzing biological samples or harvests, and mutation or gene-expression libraries for individuals with distinct or desirable properties. Most methods require taking individual colonies grown from a sample, harvest or library in the form of a microtiter dish from a petri dish, then incubating them for growth over a period of time, followed by significant sample processing and preparation. All of this is done before the analysis phase. If many individuals are to be analyzed, this step is undesirable. Analytical methods requiring less sample preparation have been developed for use in analyzing large numbers of samples.

Improvements to mass spectrometry sample analysis that allow for high throughput analysis of many samples have been described in WO 00/48004. The method purifies the components of interest using methods such as adding volatile buffers, organic solvents or ion exchange resins, thereby eliminating the commonly required column separation steps before injecting the sample into the mass spectrometer. In addition, the components could be attached to the solid support and purified. However, the method still requires some purification of the component (s) of interest to be performed prior to analysis, followed by injection of the purified sample into the mass spectrometer.

The elimination of sample component purification in the analytical method is described in US 5914245. In this method, microcolonies of cells on the bottom were optically tracked over time for changes in the optically detectable signal. Contacting the cell with an optical signal substrate generates an optically detectable signal from which an optically detectable signal is produced via an enzymatic reaction. Tracking of optically detectable signals over time makes it possible to characterize the activity of enzymes produced by the microcolonies. In this method, cells can be eluted or infiltrated to expose the optical signal substrate to the cell contents. The method eliminates sample purification steps prior to analysis, but the limitation is that the assay is based on optical detection, which is applicable only if an optical substrate having the appropriate absorbance or fluorescence properties is available for the enzyme to be analyzed. Is to make it.

In US Pat. No. 6,472,163, the method of US Pat. No. 5,914,245 is improved through the use of various types of microporous membranes which are easy to handle and have chemical resistance as substrates. Further improvements include better temperature control, more compact illumination systems, and the use of various indicators for direct and indirect or paired analysis. The limitation remains that all assays are tracked by absorbance or fluorescence techniques.

Detection of organic molecules using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) in the form of high-density array samples was described by Brown et al . ( Anal. Chem . (1999) 71: 3318-3324). In this method, a sample is injected into a silicon nanovial, which provides for the analysis of femtotor to picoliters having compounds in concentrations of x 10 ^-2 to 1 x 10 ^-4 molar, thus preventing the detection of the atomole to femtomol amounts of molecules. Make it possible. This approach is required if the only means of mapping is by rastering the main ion beam, which limits the field of view to 1000 x 1000 μm ² . Thus, while an extremely sensitive dose of ToF-SIMS detection was applied to a high throughput miniaturized assay system, only solutions containing pure compounds were analyzed. Thus the assay system as described is applicable to simple solutions that require the movement of femtor in an array of chambers before analysis. Expanding to the analysis of microbial products still requires significant sample processing to move incubation time and many complex and / or filtered solutions into the array.

ToF-SIMS analytical techniques have already been used in the analysis of various types of biological samples, all of which have the required sample preparation procedures before placing the sample into the ToF-SIMS instrument. The types of biological samples analyzed by ToF-SIMS include peptides prepared from purified proteins (Jabs and Assmann; Journal of Chromatography (1987) 414 (2): 323-333), proteins or other cellular components that bind to metal surfaces. (Michel et al., Langmuir (2002) 18 (8): 3281-3287; Sjoevall et al., Analytical Chemistry (2003) 75 (14): 3429-3434), microbial cell walls in freeze-dried preparations (Tyler et al., Proceedings of the International Conference on Secondary Ion Mass Spectrometry , 12th, (2000) pp 943-946, Editors: Benninghoven, Alfred.Publisher: Elsevier Science BV, Amsterdam, Neth.), Model Phospholipid Membrane Following Freeze-Crack (Cannon et al., Journal of the American Chemical Society (2000), 122 (4): 603-610), freeze-cracked and freeze-hydrated liposomes and red blood cells (Pacholski et al., Rapid Communications in Mass Spectrometry (1998), 12 (18: 1232- 1235), freeze-cracked and frozen hydrated paramecium (Colliyer et al., Analytical Chemistry (1997), 69 (13): 22 25-2231), and radioisotope labeled compounds (Glassgen et al., Pesticide Biochemistry and Physiology (1999), 63 (2): 97-113) in cleaved cells.

All methodologies and biological sample forms described above have limitations in analyzing a large number of samples to identify rare, preferred samples due to sample handling and / or detection procedures. The problem to be solved, therefore, is to develop a methodology that will enable the direct analysis of individual organisms without sample processing and preparation and without limitations on absorbance and fluorescence techniques.

Applicants solved the above-mentioned problems by developing a fast and effective method of applying travel-time secondary ion mass spectrometry (ToF-SIMS) to the analysis of untreated microorganisms. Using this method, the biological product of an organism that remains untreated when introduced into a ToF-SIMS instrument is analyzed by surface analysis to enable the characterization of individual organisms. The individual grows or transitions to the surface of the membrane to form an array. In addition, the array of unprocessed organisms is processed in situ and introduced directly into the ToF-SIMS device. The use of this method allows for rapid analysis of an array of mixed populations of microorganisms so that individuals with different cell contents are identified. The method thus provides a substantial advance over the available methods of analyzing large biological collections to identify individuals of interest.

Summary of the Invention

The present invention provides a method of using ToF-SIMS to detect differences in biological organisms. In one embodiment, the organism is provided on a vacuum compatible support for introduction into the ToF-SIMS device. In a second embodiment the organisms form an array on the vacuum compatible support, which organisms remain untreated when introduced into the ToF-SIMS device. In a third embodiment, an array of organisms grown in a primary medium is contacted with a secondary medium to produce a ToF-SIMS detectable product. In a fourth embodiment, an array of organisms is contacted with a substance to produce a ToF-SIMS detectable product. An array of organisms is mapped to associate ToF-SIMS data with individuals in the array. Arrays of organisms may be cloned to provide living samples that are associated with the sample analyzed.

Therefore, the present invention

a) comprising a colony of biological organisms producing a product detectable by ToF-SIMS on a vacuum compatible support;

b) performing ToF-SIMS analysis on the colonies of (a) to generate data;

c) associating the data of (b) with the colony of (a) to identify a biological organism that produces a ToF-SIMS detectable product. to provide.

In another embodiment the invention is

a) a mixed population of biological organisms present in a two-dimensional array on a vacuum compatible support, wherein at least one organism produces a product detectable by ToF-SIMS;

b) performing ToF-SIMS analysis on the organism array of (a) to generate data;

c) mapping the array to give each organism a unique locus on the array;

d) identifying one or more loci on the array in which the ToF-SIMS detectable product is present;

e) providing a method of identifying a biological organism producing a ToF-SIMS detectable product, comprising identifying the organism producing the ToF-SIMS detectable product by associating the data with the unique organism locus of (c).

Similarly, the present invention

a) a mixed population of biological organisms present in a two-dimensional array on a vacuum compatible support, wherein at least one organism produces a primary product;

b) contacting the array of (a) with a predetermined material under conditions such that the primary product reacts to produce a ToF-SIMS detectable product;

c) performing ToF-SIMS analysis on the organism array of (a) to generate data;

d) mapping the array to give each organism a unique locus on the array;

e) identifying one or more loci on the array in which the ToF-SIMS detectable product is present;

f) providing a method of identifying a biological organism producing a ToF-SIMS detectable product, comprising identifying the organism producing the ToF-SIMS detectable product by associating said data with the unique organism locus of (d).

In another embodiment the invention is

a) having a mixed population of biological organisms present in a two-dimensional array on a vacuum compatible support;

b) transferring the array to a secondary growth medium such that at least one incubated biological organism on the secondary growth medium produces a product detectable by ToF-SIMS;

c) performing ToF-SIMS analysis on the organism array of (a) to generate ToF-SIMS data;

d) mapping the array to give each organism a unique locus on the array;

e) identifying one or more loci on the array in which the ToF-SIMS detectable product is present;

f) providing a method of identifying a biological organism producing a ToF-SIMS detectable product, comprising identifying the organism producing the ToF-SIMS detectable product by associating said data with the unique organism locus of (d).

1A, 1B, 1C and 1D show white X-Gal-supplied E. coli. E. coli colonies (A, C) and blue X-Gal-supplied E. coli. Positive (A, B) and negative (C, D) ToF-SIMS spectra obtained from E. coli colonies are shown.

FIG. 2 shows the grayscale of the intensity ratio of the ToF-SIMS positive molecular secondary ionic character of the Indigo derivative to the total secondary ionic yield for each colony data set, photographed on a lattice according to the sample step coordinates of each colony. grayscale) map.

3A and 3B show X-Gal-supplied E. coli. The results of key component analysis and multivariate curve separation applied to positive and negative ToF-SIMS data combined from E. coli colonies are shown. The spatial distributions of the calculated spectra and shapes include A: general shape; And B: indigo product.

4A and B are X-Gal fed E. coli with gold Au ₁ and Au ₃ ion beams. ToF-SIMS negative secondary ion map of E. coli colonies.

5A, B and C show spatial distribution maps generated from specific peak intensities from ToF-SIMS mass spectral data in each pixel of the colony array. A: distribution of chlorine: B: [phosphate + amide / CNO] distribution of secondary ions; C: Distribution of secondary ions on bromine and indigo molecules.

Figure 6 shows E. coli fed with water (A, C) or phenylalanine (B, D) after 6 hours (A, B) or 24 hours (C, D). Negative ToF-SIMS data from E. coli is shown.

7A, B and C show petal and pET-24d E. The results of key component analysis and multivariate curve separation applied to positive and negative ToF-SIMS data obtained from 11 colonies from a mixture of E. coli are shown. A: concentration of factor 1 and factor 2 in each colony; B: calculated positive ToF-SIMS spectrum associated with factor 1, factor 2; C: Calculated negative ToF-SIMS spectrum associated with factor 1, factor 2.

8 is this. Principal component score plots generated from ToF-SIMS data of E. coli β-galactosidase / MUG samples are shown.

9 is E. Principal component score plots generated from ToF-SIMS data of E. coli β-galactosidase / NPG samples are shown.

10 is this. Results of major component analysis and multivariate curve separation applied to ToF-SIMS data from E. coli β-galactosidase + reactant samples are shown. The concentrations and calculated spectra for each of the five factors appearing from the data analysis are shown.

11A-D show spatial distribution maps generated from specific peak intensities from ToF-SIMS mass spectral data in each pixel of a colony array. Three panels consist of a phosphate map 11 A, a m / z 279 / m / z 281 ratio map 11B from a mixture of two different colony types (Q, Q + 4), and (m The comparison of the map 11C with the / z 281) / (m / z 279) ratio is shown. FIG. 11D shows the negative secondary ion spectra from the pixels that make up two different colony types (Q, Q + 4) labeled "A" and "B" on the map of FIG. 11A.

12 shows map locations and spectra of Principal Component 1 and Principal Component 2 from ToF-SIMS data analysis from Yarrowia colonies.

Figure 13 shows negative ToF-SIMS data: Q + 4 colonies grown in Supor® and Q + 4 colonies on Sufor® after performing the saponification process.

The next sequence is 37 C.F.R. 1.821-1.825 ("Requirements for Patent Applications Containing Nucleotide Sequences and / or Amino Acid Sequence Disclosures-the Sequence Rules"), and comply with the International Intellectual Property Organization (WIPO) Standard ST.25 (1998) and EPO and PCT (Rule 5.2 and 49.5 (a-bis), and the sequence listing requirements in section 208 of the Administrative Guidance and Annex C). The symbols and formats used for nucleotide and amino acid sequence data are 37 C.F.R. Complies with the provisions of Sec. 1.822.

SEQ ID NO: 1 shows a Q + 4 Yarrowy liposome for the expression of four genes: Mortierella alpina high affinity PUFA elongase , M. alpina Δ5 desaturase, Saprolegnia diclina Δ17 desaturase and M. alpina Δ6 desaturase. 10.3 kb DNA fragment of the tikka strain.

The present invention describes a method for analyzing the contents of a microorganism using ToF-SIMS data collection and analysis, wherein the cells to be analyzed do not require pretreatment and are untreated when introduced into a ToF-SIMS instrument. The present invention also describes ToF-SIMS analysis of microbial colonies grown on vacuum compatible membranes that are directly transferred to ToF-SIMS instruments. Colonies exist as arrays on the membrane and one aspect of ToF-SIMS data is used to map an array of colonies. Another aspect of ToF-SIMS data is used to identify cell content differences between colonies in arrays on membranes. It is then possible to obtain a colony of an individual with desired or distinguished cell contents, in which replicating the colony array in advance. By this method a large number of individual colonies, such as colonies present in biological samples or harvests, and mutant or gene-expression libraries can be analyzed quickly. The following definitions may be used to describe the claims and the specification.

"Move-time secondary ion mass spectrometry" is abbreviated as ToF-SIMS.

The term "ToF-SIMS detectable product" means any compound or substance that can be detected by a transit-time secondary ion mass spectrometry detection method. The ToF-SIMS detectable product may be a specific known compound derived from a reactant provided or from a reactant present in a cell. In addition, the ToF-SIMS detectable product may be one which, although not defined, causes a difference in ToF-SIMS data between the samples being compared.

"ToF-SIMS analysis" means a method applied by a ToF-SIMS device to collect data from a sample.

"ToF-SIMS analysis" includes the collection of data from a ToF-SIMS device as well as processing the data to generate information characterizing the sample being analyzed. ToF-SIMS analysis can include principal component analysis (PCA) and multivariate curve separation (MCR).

A "mixed population" or "mixed population of cells" is a population of biological cells, including several kinds of individuals. Individuals are of a different kind due to differences in cell contents. Mixed populations may include various strains of organisms, mutant populations, or other populations that differ between individuals.

The term "array" when used with respect to the orientation of a biological organism on a substrate refers to a two-dimensional distribution of the organism in which the spatial orientation of the organism is maintained.

By "raster" is meant to manipulate the main ion beam of the ToF-SIMS instrument with respect to the pixel array over the area of the sample, or to move the sample under the (fixed) main ion beam according to the pixel array over the area of the sample.

The term "vacuum compatible support" means a surface or membrane composed of a material that is intact and capable of withstanding very low air pressures. In addition, the vacuum compatible support must be a membrane that can be placed in such a way that the array of biological organisms is maintained in the spatial integrity of the organism. Suitable materials include, but are not limited to, paper materials and polymeric materials such as nylon, nitrocellulose, polyethersulfones, polysulfones, polycarbonates, polystyrenes and silicon / silica and glassy carbon.

The "aspect" of the ToF-SIMS data is itself part of the overall ToF-SIMS data providing a set of information that can be used to characterize the sample being analyzed.

"Mapping an array" means giving each object in the array a defined position. Defined locations, also called loci, can be illustrated. ToF-SIMS data can be associated with each locus, so can also be associated with each individual in the mapped array.

"Chemometric method" means a mathematical or statistical method used to correlate a measurement performed on a chemical system with the state of the system. Chemical measurement methods are used to solve complex sets of data in order to enable the characterization of the sample from which the data is collected. Examples of chemical measurement methods include principal component analysis (PCA) and multivariate curve separation (MCR).

A "major product" is a substance that can itself be modified or that can transform a reactant into a ToF-SIMS detectable product. The main product can be modified by contact with another substance, such as modification of cellular components by acid or base hydrolysis. The main product, which is an enzyme, can act on the reactants to produce a ToF-SIMS detectable product.

The term "fatty acid" means a long chain fatty acid (alkanoic acid) having a variable chain length of about C ₁₂ to C ₂₂ (although acids with longer or shorter chain-lengths are also known). Main chain length is between C ₁₆ and C ₂₂ . The structure of a fatty acid is represented by a simple representation system of "X: Y" (X is the total number of C atoms and Y is the number of double bonds).

The term "chimeric gene" refers to 1) a DNA sequence comprising regulatory and coding sequences that are not found together in nature; 2) sequences encoding portions of a protein that are not naturally contiguous; Or 3) any gene containing portions of a promoter that are not naturally contiguous. Thus, the chimeric gene may comprise regulatory sequences and coding sequences derived from different sources or may comprise regulatory sequences and coding sequences derived from the same source but arranged in a different manner than those found in nature. "Transformation" means that the foreign gene (s) are transferred within the genome of the host organism.

"Promoter" means a nucleotide sequence, typically upstream (5 ') of the coding sequence, which controls the expression of the coding sequence by recognizing RNA polymerase and other factors necessary for proper transcription. A "promoter" includes a minimal promoter, a short DNA sequence consisting of a TATA-box and other sequences that act to specify transcription initiation sites, with regulatory factors added to control expression. "Promoter" also refers to a nucleotide sequence comprising a minimal promoter and regulatory factors capable of regulating the expression of mRNA or functional RNA.

"Transfer terminator" means a 3 'non-coding regulatory sequence located downstream of a coding sequence. It includes polyadenylation recognition sequences and other sequences that encode regulatory signals that can affect mRNA processing or gene expression. The polyadenylation signal is typically characterized by affecting the addition of the polyadenylic acid pathway to the 3 'end of the mRNA precursor. The 3 ′ region may affect transcription, RNA processing or stability, or translation of related coding sequences (eg, for target genes, etc.).

Standard recombinant DNA and molecular cloning techniques used herein are known in the art and described in Sambrook, J., Fritsch, EF and Maniatis, T., Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor Laboratory Press Cold Spring Harbor, NY (1989) (hereinafter referred to as "Maniatis"); And Silhavy, TJ, Bennan, ML and Enquist, LW, Experiments with Gene Fusions , Cold Spring Harbor Laboratory Cold Press Spring Harbor, NY (1984); And Ausubel, FM et al., Current Protocols in Molecular Biology , published by Greene Publishing Assoc. and Wiley-Interscience (1987).

Travel-Time Secondary Ion Mass Spectrometry (ToF-SIMS)

In the ToF-SIMS process, a high-energy (8-25 kV), low-current (nanoamp DC, with picoamp pulse) pulsed ion beam hits the surface of the material. Secondary ions with positive and negative charges are produced from the top monolayer (10-20 μs). The secondary ions are analyzed and detected by a mass spectrometer. Mass spectra yield information about surface compositions and structures. The present invention is the first application of the surface price analysis for cells that are untreated when placed in a ToF-SIMS instrument.

In the "static" SIMS regime, the low main ion beam current and random beam raster ensure that the undamaged surface is sampled over the acquisition time. This is in contrast to "dynamic" SIMS, where the main ion beam is used to sputter / remove material from the surface and data is obtained as a function of depth. In static SIMS, secondary ions of high-mass molecules can be detected, but in dynamic SIMS, molecular information is compromised so that only elemental or low-mass information is available. The static SIMS regime is most useful for the present invention because the high-mass molecular information obtained can identify many products in the cell and the difference can be used to distinguish cells.

The types of mass spectrometers that have been used for secondary ion analysis and detection include quadrupole mass spectrometers, magnetic sector mass spectrometers, and travel-time mass spectrometers. Quadrupole mass spectrometers are limited by mass ranges (typically less than 2000 m / z) and mass resolutions (typically around unit mass resolution). Magnetic sector mass spectrometers provide very high mass resolutions, but require specific control over the mass range of interest. Both quadrupole and magnetic sector analyzer optics must be scanned to scan over the mass range. Travel-time mass spectrometers provide higher permeation of mass-independent secondary ions and substantially parallel detection of mass since the entire mass range is collected over a microsecond time. Ions are separated by the time needed to traverse the field-free path of travel that depends on mass. For static SIMS analysis of organic materials, where analysis time must be short to limit ion beam damage, a travel-time analyzer is the analyzer of choice.

Using only the travel-time principle results in a normal mass resolution of only about 500 (m / dm). This is because secondary ions are released from the sample with a significant energy distribution. Currently two different travel-time analyzer geometries are used for SIMS using various methods of energy focus. These are known as ion reflector types and triple-focus sector types. The embodiment included herein uses an ion reflector type ToF-SIMS device, but triple-focus sector devices are expected to provide similar results and thus can be used in the practice of the present invention. For both types of travel-time analyzers, the polarity of the extractor must be switched to collect secondary ions with positive or negative charge. Thus, as separate results, the "positive ToF-SIMS" spectrum meaning the signal from the positively charged secondary ions and the "negative ToF-SIMS" spectrum meaning the signal from the negatively charged secondary ions Is collected.

As seen in the films used in the present invention, one problem arising in ToF-SIMS analysis of insulating materials such as ceramics or polymers is to charge the sample. The main ion beam typically has a positive charge and the insulating sample will also immediately acquire a positive charge. If not compensated, this charge will affect the extraction of secondary ions into the analyzer. An electron gun is used to compensate for the charge buildup on the sample.

If a travel-time mass spectrometer is used, the main ion beam must be pulsed to determine the departure time. A typical mandatory cycle for the analysis of an insulated sample begins with the main ion beam pulse, followed by the time that the secondary ion extraction voltage turns on. Turn off the extraction voltage, then turn on the ejection gun. After the time during which the highest mass secondary ion of interest reaches the detector (typically 100-200 microseconds), another compulsory cycle begins.

The main ion sources used for ToF-SIMS included inert gases (argon, xenon), cesium, oxygen, silicon penta- and hexa-fluoride, and so-called "liquid metals" such as gallium, indium and gold. For mapping experiments where the main ion beam is rasterized, a liquid metal ion source is required because of the strength / luminance of the ion source even when focused to sub-micron size. Gold ion sources have recently been developed that provide a mapping capability with an improved high-mass secondary ion signal because the signal increases with the mass of the main ion. Au-1, Au-2 and Au-3 can be obtained as the ion source. New major ion sources, including new multi-atom major ion sources, continue to be developed. Although any usable ion source is applicable to the present invention, an ion source using cesium, gallium or Au-1 as the main ion source is preferred.

ToF-SIMS is a semi-quantitative technique. Since the physical phenomena of ionization and desorption are paired in the present invention, the yield of secondary ions depends on the chemical environment or the so-called "matrix effect". However, semi-quantitative or relative information (“more” to “less” or related to the measured / final-use properties) is available from a series of related samples through the use of peak area ratios. The area under the peak of interest (eg, molecular secondary ions of known additives or products) is expressed as the ratio to the area under the peak selected to serve as a reference. For example, the relative measurement of silicon oil contamination on the Mylar® PET film surface was determined by the peak area of the silicone oil at m / z 147 (C3H9SiO-C2H6Si +) versus a series of Mila ^* PET samples. Can be obtained by comparing the ratio to the PET peak area at m / z 104 (C6H4CO +). If an apparent reference peak is present or unknown, some can be used as reference for the total yield of positive or negative secondary ions or for the same spectrum of the same. Such methods using the ratios in the ToF-SIMS data as a criterion for distinguishing samples with different properties are known to those skilled in the art and span a large number of colonies where the ratio of the secondary ion total yield of the amount of indigo molecular ion area is analyzed. It is useful in practicing the present invention as shown in Example 2 which is used as a relative measure of indigo production.

ToF-SIMS data can be better evaluated using chemical measurements. See B.M. Wise and N.B. As defined by Gallagher, PLSToolbox reference manual (PLS_Toolbox Version 2.0, copyright 1998, Eigenvector Research, Inc., Manson, WA, page 31)], "Chemical measurements can be applied to chemical systems through the application of mathematical or statistical methods. It is the science that relates the measurements performed to the state of the system. " ToF-SIMS data is multivariate; "Multivariate" means data consisting of measurements of multiple variables per sample. In the case of ToF-SIMS mapping data, each mass channel can be considered a variable, and the intensity for each of the plurality of variables is collected for each pixel of the mapping array. Another meaning of the multivariate nature of ToF-SIMS is the presence of multiple spectral patterns from different species that make up the overall positive or negative ToF-SIMS spectrum.

Two factors inherent in the SIMS technique use multivariate statistical techniques known to those skilled in the art, such as principal component analysis (PCA) and multivariate curve separation (MCR), which aid in the interpretation and extension of SIMS data. One factor is that unlike other analytical techniques, including mass spectrometry such as GC-MS (gas-chromatography-mass spectrometry), there is no separation of species before secondary ions enter mass spectrometry. Mass spectral data is a tangle of secondary ion patterns from all species coexisting on the surface, and many low-mass hydrocarbon peaks are common or common to many species. The second factor is that in mapping experiments where the main ion beam is rasterized over the area of the surface, the concentration of current at the concentrated spot results in higher occurrence of charge and beam damage. The final result is a much lower signal / noise for mass spectral data obtained in this mode.

In a typical mapping experiment, ToF-SIMS spectral data is obtained at each pixel of a 256 x 256 pixel array across the analysis area. Principal component analysis takes the 65536 spectra of the set and first attempts to find out what constitutes a variance in the data, and then reclassifies the data into dominant components that continue to account for less of the variance. In doing this, the PCA mainly brings together pixels with similar spectral patterns. As noted above, both positive ToF-SIMS and negative ToF-SIMS mapping data can be obtained in separate data processing. Negative and positive mapping data may be analyzed separately by principal component analysis, or as one combined set of spectra where pixel records between positive and negative data can be obtained.

The results of key component analysis depend on the preprocessing of the data. It is common practice for those skilled in the art to use mean-centered data. In mean-centering, the mean for each column representing the intensity in one particular mass channel for all pixels is subtracted from each item of that column. In addition, for all the embodiments described herein using principal component analysis, a series of pixel spectra (65536 for a 256 x 256 array) were normalized prior to mean-centering. This generally means normalizing the total ion yield or a subset of the total ion yield as shown in the examples.

Multivariate curve separation is a method of separating data that is a complex of many causal factors into their individual factors. The results of the PCA can be used as a starting point for the MCR to further reduce the ToF-SIMS data to specific species. Those skilled in the art will be familiar with the techniques as described in the PLS_Toolbox tutorial (PLS_Toolbox, Version 2.0, Eigenvector Research, Inc., Manson, WA) and will be able to implement them for use in the present invention.

ToF-SIMS Detectable Product

The nature of the ToF-SIMS technique requires several conditions that must be met for the species to be detected:

ToF-SIMS is performed at ultra-high vacuum (<1 x 10 ^-9 torr pressure). The species to be detected must therefore be vacuum-compatible.

The compound of interest must be present in itself at the outermost 10-20 mm of the sample being analyzed.

The compound must produce a secondary ion peak or spectral pattern that distinguishes it from the secondary ion signal generated from all other species that contribute to the full spectrum.

There are a number of substances that can be detected by ToF-SIMS within these limits. Biological substances that can be detected include many components of living cells. Some examples include, but are not limited to, fatty acids, proteins, carbohydrates, and organic acids. Fatty acids are preferred compounds for detection using the methods of the invention and include lauric acid, myristic acid, palmitic acid or stearic acid; Oleic acid, linoleic acid, linolenic acid, di-homo-gamma linoleic acid, arachidonic acid, stearic acid, eicosaterateic acid, eicosapentaenoic acid, docosahexanoic acid, hydroxy fatty acids, peroxy fatty acids, branched chain fatty acids and phospholipids And compounds containing substituent fatty acids such as phospholipid fragments, triglycerides and triglyceride fragments.

Certain cellular components may not be readily detectable in ToF-SIMS in their original state, but may be converted to ToF-SIMS detectable products through reactions mediated by externally supplied materials. For example, acid or base treatment can be readily applied to cells to convert some cellular components. One example is saponification of triacylglycerides and phospholipids using sodium hydroxide. Methanol containing potassium hydroxide is a common treatment for saponification. The use of potassium hydroxide alone is the preferred saponification treatment for use in the present invention. Such treatments that can be applied to colonies on the membrane where the integrity of the colonies are maintained can be used in the practice of the present invention.

ToF-SIMS detectable products can also be made by living cells from the reactants supplied to the cells. The reactants may be natural substances such as amino acids or various sugars, or the reactants may be specially synthesized compounds designed to be converted by specific cellular processes. For example, X-Gal (Holt, SJ and PW Sadler, Proc. Royal Sco. (London) 148B: 495 (1958)) is designed to be converted into a product visually detectable by the β-galactosidase enzyme. . However, ToF-SIMS allows detection of many substances as described above, which are not visually detectable.

Travel-time analyzers theoretically have no mass limit. However, since desorption from the surface is part of the treatment, there is a finite radius of the influence of the main ion beam that limits the size, and therefore there is a mass of secondary ions that can be desorbed. Thus, ToF-SIMS detectable products are generally 8 kD or less.

Organisms for ToF-SIMS Analysis

The organisms to be analyzed using ToF-SIMS in the present invention include any eukaryotic and prokaryotic organisms that can be applied to the membrane support and incorporated into the ToF-SIMS instrument. Preferably, the organisms are applied to the membrane support so that the individuals are separated from each other. And the organism to grow as a colony particularly suitable in the practice of the invention, the saccharide to my process (Saccharomyces), a species of yeast, such as Pichia (Picchia) and Yarrow baby.- (Yarrowia); Rhodococcus (Rhodococcus), Streptomyces (Streptomyces), evil Martino fine test (Actinomycetes), Corynebacterium (Corynebacterium), Bacillus (Bacillus), Escherichia coli (Escherichia coli), Pseudomonas (Pseudomonas), Salmonella ( Gram-negative and gram-positive bacteria such as Salmonella ) and Erwinia . Fungi such as Penny rooms Solarium (Penicillium), Fusarium (Fusarium), Aspergillus across Scotland (Aspergillus), grape Spokane LA (Podospora), Creative Source Four Leeum (Chrysosporium), Trichoderma (Trichoderma) and Neuro Castello La (Neurospora) It may also be included. Cells derived from eukaryotic organisms, including mammalian cell lines and major cultures, and plant cells such as corn, rice, wheat, soybean, tobacco and arabidopsis, which can grow in culture according to the above criteria, It may be used for implementation.

Samples of organisms for analysis can be from environmental, clinical, food, experimental or other sources. The ToF-SIMS method of the present invention can be used to identify contaminants or pathogens present in a sample based on ToF-SIMS detectable distinct components present in contaminant or pathogen cells. Preferably the fatty acid component detected by ToF-SIMS is used to identify bacterial species in which distinct fatty acids are present.

Vacuum compatibility support

The vacuum environment and surface specificity of the ToF-SIMS technology are important considerations in the selection of vacuum compatible supports (such as membranes) required for the practice of the present invention. The membrane should be suitable for low pressure vacuum environments and free of surfactants or other surface-shifting agents or additives. The membrane is also preferred if it is porous to support the growth of the organism and to support the growth of the organism when placed on the surface of the culture medium. Any membrane may be used, such as a membrane of polyethersulfone, polysulfone, nylon, polycarbonate or nitrocellulose (washed with solvent to remove the surfactant) that meets the above requirements. Colonies may be transferred from growth media or growth supporting films onto other types of films, such as silicon wafers or glassy carbon plates, suitable for ToF-SIMS devices. Silicon wafers should be considered conductive with low resistance, and the orientation or dopant thickness may vary, such as with boron or phosphorus and with 110 or 111 orientation.

Cloned organism samples

Colonies of organisms grown on the surface of the medium can be replicated while maintaining their original spatial orientation. A filter or membrane placed on top of the colonies on the surface will take some cells from each original colony. When a second filter or membrane is placed on the growth medium and incubated under growth conditions, the cells divide and form a replica of the original colony set. Any number of copies can be made from the original set of colonies or from any subsequent replicated set. Colonies can be replicated in the same way from one filter or membrane to another. Replication of one set of organisms allows one set to be maintained, while another set is analyzed in a destructive manner. As long as the orientation of the colony sets maintained and analyzed can be aligned, individuals identified as having specific properties from the assay can be propagated.

Individual organisms in mixed populations

Colony growth of organisms on the surface constitutes a spatial distribution or array of individuals. If colonies grow on a filter or membrane support, they can transition while maintaining their spatial distribution. Transitioning to other media, in solution, and into experimental instruments is both useful for the practice of the present invention.

Maintaining an array of individuals within a mixed population allows for the identification of specific individuals with different characteristics. Mixed populations can be composed of individuals that are nearly identical in character, with one or several different individuals. Mixed populations may include different individuals with multiple differences in characteristics.

Population analysis

Analysis of mixed populations to identify individuals with desirable characteristics is used in programs involving mutagenesis, gene shuffling, or other methods of altering the genetic structure of an organism. Large mixed populations containing genetically altered individuals are commonly referred to as libraries. Analyzing thousands or even tens of thousands of objects in a library may be necessary to identify several individuals with the desired characteristics. The method of the present invention allows for rapid and efficient analysis of individuals directly on the membrane on which they grow. In one embodiment the individuals on the membrane are analyzed directly without the need to transfer to microtiter plates or undergo other processing steps. In another embodiment a processing step is used that can be applied directly to the membrane and does not destroy colony integrity. The present invention shows for the first time that ToF-SIMS, a surface analysis technique, can identify cellular components directly from colonies grown on membrane supports.

The invention is defined in more detail in the following examples. It is to be understood that these examples represent preferred embodiments of the invention but are given by way of example only. From the above discussion and these examples, those skilled in the art will be able to ascertain the main features of the present invention, and various changes and modifications may be made to the present invention to adapt the present invention to various uses and conditions without departing from the spirit and scope thereof. have.

General method

Standard recombinant DNA and molecular cloning techniques used in the examples are known in the art and described in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual ; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, (1989) (Maniatis) and TJ Silhavy, ML Bennan, and LW Enquist, Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1984) and Ausubel, FM, etc. , Current Protocols in Molecular Biology, pub. by Greene Publishing Assoc. and Wiley-Interscience (1987).

Materials and methods suitable for the maintenance and growth of bacterial cultures are known in the art. Techniques suitable for use in the following examples are described in Manual of Methods for General Bacteriology (Phillipp Gerhardt, RGE Murray, Ralph N. Costilow, Eugene W. Nester, Willis A. Wood, Noel R. Krieg and G. Briggs Phillips, eds), American Society for Microbiology, Washington, DC. (1994) or Thomas D. Brock in Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells are Aldrich Chemicals (Moldwaukee, WI), DIFCO Laboratories, Detroit, MI unless otherwise specified. ), Gibco / Bial (GIBCO / BRL, Gaithersburg, MD) or Sigma Chemical Company, St. Louis, Mo.

The abbreviations mean the following: "sec" means seconds, "min" means minutes, "hr" means hours, "d" means days, and "μL" means microliters "ML" means milliliters, "L" means liters, "μM" indicates micromolar concentrations, "mM" indicates millimolar concentrations, "M" indicates molar concentrations, and "mmol" indicates millimoles Where "μmole" is micromolar, "g" is grams, "mg" is milligrams, "μg" is micrograms, "ng" is nanograms, "U" is units, "μm" For micrometers, "cm" for centimeters, "mm" for milliliters, "RPM" for revolutions per minute, "bp" for base pairs, "kb" for kilobase and m / z means mass to charge ratio.

Example 1

Lee through indigo detection. ToF-SIMS Identification of E. coli-expressing β-galactosidase

This example shows the ability of ToF-SIMS to distinguish bacterial colonies expressing β-galactosidase enzyme from those that are not. It uses a system with obvious visual changes in organisms that can convert the reactants (colorless 5-bromo-4-chloro-3-indolyl-β-D-galactoside) into products (blue indigo derivatives). This allows for immediate verification of ToF-SIMS data. This example also shows a method for transferring the harvest of bacterial colonies from the solid growth medium to the high-vacuum analysis chamber of the ToF-SIMS instrument while maintaining the spatial relationship of the colonies.

Test system:

The Escherichia coli LacZ gene encoding β-galactosidase (β-gal) is a classic histochemical reporter gene (Beckwith, JR Lac: The genetic system.In The Operon . JH Miller and WS Reznikoff, Eds. Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1980)). Its activity can be detected using a variety of substrates, all of which have galactose bound to residues whose properties change upon release from galactose through β-D-glucoside binding (Wallenfels, K., and R. Weil In The Enzymes.PD Boyer, Ed. 3 ^rd ed.Academic: NY, 7: 617 (1972). One useful chromogenic substrate that produces a precipitated product upon cleavage by β-gal is an indole derivative, 5-bromo-4-chloro-3-indolyl-β-D-galactosid (or “X-gal” Holt, SJ, and PW Sadler. Proc. Royal Soc . (London) 148B: 495 (1958)). When β-gal cleaves the X-gal glycosidic bond, a soluble colorless, doxolic monomer is produced. Two of the liberated doxyl residues then form a dimer, which is non-enzymatically oxidized to yield a halogenated indigo, a blue compound that is very stable and insoluble. Thus, when cells are grown in the presence of X-gal, they may or may not express lacZ, respectively, based on the presence or absence of indigo products. It is possible to fractionate E. coli cells (commonly known as "blue / white" assays). Structurally, this. It is important to recognize that X-gal indigo product is maintained in E. coli cells and therefore the product is not excreted through the cell membrane into the surrounding environment.

this. Preparation and Growth of Nylon Coli Colonies on Nylon Membranes:

30 μL of aqueous 100 mM isopropylthiogalactoside (IPTG) and 20 mg / mL X-gal (5-bromo-4) in a Petri dish containing ˜20 mL of LB agar with 100 μg / mL ampicillin 50 μL of a dimethylformamide solution of -chloro-3-indolyl-β-D-galactoside) was placed on top and the plate was left open for 15 minutes to dry the surface. A nylon 66 membrane cast on a polyester support of 0.45 μm pore size (Boehringer Mannheim, Mannheim, Germany), with a circular microporous positive charge, was placed on the surface of the agar and the E. coli in pUC18. E. transformed with a library of E. coli K12 genomic DNA. Cultures of E. coli DH5α were spread over the nylon membrane. The culture is such that 34% of the plasmids have DNA inserts and thus 34% of colonies grown from the cultures are composed of populations that do not express β-galactosidase due to the disruption of the lacZ gene in pUC18 by genomic DNA inserts. Predetermined (Beckwith, JR Lac: The genetic system.In The Operon . JH Miller and WS Reznikoff, Eds. Cold Spring Harbor Laboratory: Cold Spring Harbor, NY (1980)). After incubation overnight at 37 ° C., the nylon membrane was covered with ˜1500 colonies, 34% of which was white and the rest were blue. The blue colony contained bacteria expressing β-galactosidase and thus could convert X-gal into indigo derivatives that were easily visualized (Holt, SJ, and PW Sadler, Proc. Royal Soc. (London) 148B 495 (1958).

Introduction of Colonies into Vacuum Chambers for ToF-SIMS Analysis

A 1 cm ² portion of the nylon membrane was cut, peeled off from the growth medium, and placed on a stainless steel round sample holder (called "puck") by screwing the molybdenum mask onto the membrane. The sample was then introduced into a PHI Model 7200 ToF-SIMS instrument (Physical Electronics, Eden Prairie, MN) according to the manufacturer's instructions. After depressurization was carried out in a pre-chamber at atmospheric pressure to <1 x 10 ^-5 Torr, the sample stage was pushed using a magnet moving rod to the main analytical chamber with a pressure of <1 x 10 ^-7 Torr.

Confirmation of ToF-SIMS detection of blue to white colonies based on spectroscopic differences

The blue and white colonies were analyzed using the following ToF-SIMS conditions: Cesium main ion source, pulsed electron ejection gun for charge compensation, and ion reflector type spectrometer. The area of each colony collected was 200 × 200 μm ² . ToF-SIMS spectroscopic differences were detected between the two colonies. For blue colonies, the ToF-SIMS data shows positive and negative charges at m / z 486 (FIG. 1B, D), the size of the protonated and deprotonated molecular ions of the indigo derivatives, respectively. Clusters of each of the secondary ions are shown. The white colonies did not show secondary ion clusters with positive or negative charge at m / z 486 (FIGS. 1A, C). That is, the ability of ToF-SIMS to identify biological organisms that produce indole derivatives of X-Gal, which are specific ToF-SIMS detectable products, has been demonstrated.

Example 2

Different Methods of ToF-SIMS Analysis of Random-arrayed Bacterial Colonies

This example comprises 1) acquisition of single-point spectra from known colony locations; 2) mapping across regions containing multiple colonies by rastering the main ion beam; And 3) three different methods for analyzing random-arrayed bacterial colonies based on mapping across regions containing multiple colonies by rastering the sample stage under the main ion beam. Each methodology is used to generate and not produce ToF-SIMS detectable products (eg, indole derivatives of X-Gal). Coli colonies were identified in the array.

Colony Analysis with Single-Point ToF-SIMS Analysis

As described in Example 1, E. coli comprising blue colonies expressing β-galactosidase and white colonies not expressing β-galactosidase. Nylon membranes were prepared covered with a mixture of E. coli colonies. Samples of the membrane were transferred to a ToF-SIMS instrument as described in Example 1.

Sample stage coordinates for each of the 12 colonies (mix of blue and white colonies) were determined and recorded by visual inspection of the membrane through the eyepiece of the ToF-SIMS instrument, aligned on a micrometer scale. The sample stage was moved back to the set of angular coordinates and the 200 × 200 μm ² area was analyzed separately for each colony using the ToF-SIMS conditions described in Example 1.

The intensity of the secondary ionic character on the positive molecular indigo derivatives (as described in Example 1 and shown in FIG. 1) is shown as the ratio to the total secondary ion yield for each colony data set. The ratio was translated into a grayscale colormap and plotted on the grid according to the sample stage coordinates of each colony. The colormap of ToF-SIMS data in the form of colony arrays showed six dark white points, one less dark white point, and five barely recognizable gray points (FIG. 2). As evidenced by the visual color of the colonies, which are blue and white, respectively, seven white dots represent the stage positions of seven colonies containing indigo derivatives, while five gray dots represent colonies containing no indigo derivatives.

Thus, the single-point of the ToF-SIMS analysis technique is useful for the identification of biological organisms that make specific ToF-SIMS detectable products.

Colony analysis via ToF-SIMS secondary ion mapping through key ion beam rastering and chemical measurement data reduction

As described in Example 1, E. coli comprising blue colonies expressing β-galactosidase and white colonies not expressing β-galactosidase. Nylon membranes were prepared covered with a mixture of E. coli colonies. Samples of the membrane were transferred to a ToF-SIMS instrument as described in Example 1.

ToF-SIMS analysis was performed on a 400 × 400 μm ² area of the membrane comprising one blue colony portion and one white colony portion. Mass spectrometers with gallium main ion beam and ion reflector designs with pulsed electron ejection guns for charge compensation were rastered at regular spatial intervals over an analysis area with 256 x 256 pixel resolution. In each pixel, an array over the analysis area was constructed to obtain a full surface mass spectrum.

The total secondary ion signal obtained by rastering the main ion source was lower than that obtained using the method described in Example 1, and thus, the chemical measurement data reduction method was used to improve the spectral contrast. The datafile was first taken from 256 x 256 pixels to 204 x 256 pixels (320 x 400 μm ² ) so that it contained almost the same parts of both colonies. The resulting set of 52,224 combined positive and negative ToF-SIMS pixel spectra were first normalized (from total positive secondary ion yield for positive set and m / z 50 to negative total future negative secondary ion yield). . Next, the data set is averaged-centered and input for principal component analysis (PCA) (PLS_Toolbox Version 2.0, Eigenvector Research, Inc., Manson, WA) associated with MATLAB Version 5.3, The Mathworks, Inc., Natick, MA. Used as data. The output of the analysis was then fed to a multivariate curve separation routine (PLS_Toolbox, MATLAB). The process, familiar to those skilled in the art, classifies pixels into similar spectral patterns and then separates them into spectral components. Two factors appeared. Spatial distribution maps were created by plotting the intensity or "concentration" of each factor per pixel. One map showed the spatial distribution of spectral characteristics common to both colonies (FIG. 3A). The map was accompanied by a calculated spectrum for the common characteristic. The second map (FIG. 3B) showed the spatial distribution of spectral characteristics unique to one of the colonies. The unique spectral properties were peaks corresponding to bromine and indigo molecular secondary ions present in colonies expressing β-galactosidase. It was its blue color to demonstrate that colonies with these properties express β-galactosidase.

Blue colonies and β-galactosidas expressing β-galactosidase as described in Example 1 except that the membrane was a Supor® polyethersulfone membrane (Pall-Gellman, Ann Arbor, MI). E. coli containing white colonies that do not express an aze. A membrane covered with a mixture of coli colonies was prepared.

Au ₁ gold main ion beam and Au ₃ using a mass spectrometer with pulsed electron ejection gun for charge compensation and with ion reflector design (Ion-ToF Model ToF IV, Ion-ToF GmbH, Muenster, Germany) ToF-SIMS analysis was performed on a Supor® membrane of 500 x 500 μm ² area containing one blue colony by a primary ion beam raster using a gold primary ion beam. No chemical measurement data reduction was performed on the data. Negative ToF-SIMS secondary ion maps from gold ion beam data were able to detect indigo directly as shown in FIGS. 4A and B for Au ₁ and Au ₃ respectively. As shown in the figure, bromine was also detected.

Thus mapping through key ion beam rastering and chemimetric data reduction has been a useful ToF-SIMS analysis technique for the identification of biological organisms making specific ToF-SIMS detectable products.

Colony analysis via ToF-SIMS secondary ion mapping through stage raster under main ion beam

Blue colonies and β-galactosidas expressing β-galactosidase as described in Example 1 except that the membrane was a Supor® polyethersulfone membrane (Pall-Gellman, Ann Arbor, MI). E. coli containing white colonies that do not express an aze. A membrane covered with a mixture of coli colonies was prepared. Samples of the membrane were transferred to a ToF-SIMS instrument as described in Example 1.

Using a mass spectrometer (Ion-ToF Model IV, Ion-ToF GmbH, Muenster, Germany) with pulsed electron ejection gun and ion reflector design for charge compensation, it was possible to raster the sample stage under a gold main ion beam to achieve 256 x ToF-SIMS analysis was performed on a 20 × 20 mm ² area film containing multiple colonies with 256 pixels. At each interval or pixel, the entire surface mass spectrum was obtained by constructing the array over the area. Next, a spatial distribution map was created using the intensity of a particular peak from the mass spectral data at each pixel. Specifically, the distribution of chlorine to detect the membrane support was used to make a pictorial map of all colonies on the array as shown in FIG. 5A. The intensity of [phosphate + amide / CNO] secondary ions per pixel was also used to indicate the location of all colonies as shown in FIG. 5B. The distribution of secondary ions on bromine and indigo molecules was used to identify the locations of the colonies that could convert X-Gal to indigo as shown in FIG. 5C. Color overlays of chlorine (blue) + [phosphate + CNO] (red) or chlorine (yellow) + [bromine + indigo] (blue) were used to easily identify the type of each colony. Comparing the secondary ion map with optical photographs of colonies, the blue color demonstrated the identification of colonies showing bromine and indigo molecular secondary ions.

Thus, mapping through stage rasters under the main ion beam was also a useful ToF-SIMS analysis technique for the identification of biological organisms that make certain ToF-SIMS detectable products.

Example 3

Phenylalanine: E. coli expressing ammonia degrading enzyme. E. coli ToF-SIMS detection:

This example shows ToF-SIMS detection in systems where the product is a water soluble low molecular weight organic acid, with no visual change in the organism capable of converting the reactants into products. This example also shows how the colonies that can produce the product can be spectroscopically distinguished even if no molecular secondary ions specific to the product are detected.

Phenylalanine: E. coli expressing ammonia degrading enzyme. Coli:

E. coli expressing enzyme activity that is not readily detectable, such as β-galactosidase. To demonstrate ToF-SIMS detection of E. coli, we used phenylalanine: ammonia degrading enzyme (PAL) from mutated Rhodosporidium toruloides as described in US6368837 . The enzyme had improved tyrosine: ammonia degrading enzyme (TAL) activity but only PAL activity was analyzed in this example.

Phenylalanine ammonia-degrading enzyme (PAL) (EC 4.3.1.5) is used for plants (Koukol et al. , J. Biol. Chem. 236: 2692-2698 (1961)), fungi (Bandoni et al. , Phytochemistry 7: 205-207 (1968) ), Yeast (Ogata et al. , Agric. Biol. Chem. 31: 200-206 (1967)) and Streptomyces (Emes et al. , Can. J. Biochem. 48: 613-622 (1970)), , Escherichia coli or mammalian cells (Hanson and Havir in The Enzymes , 3rd ed .; Boyer, P., Ed .; Academic: New York, 1967; pp 75-167). PAL is the first enzyme of phenylpropanoid metabolism and catalyzes the removal of (pro-3S) -hydrogen and -NH3 + from L-phenylalanine to form trans-cinnamic acid. this. Two strains of E. coli were compared in this example: E. coli transformed with the pETAL construct, a pET-24d plasmid containing the coding region for the EP18Km-6 mutant of the Rhodosporidium toluloides PAL enzyme (US6368837). E. coli BL21 (DE3) (Novagen, Madison, Wisconsin); And E. transformed with pET-24d plasmid. E. coli BL21 (DE3) from Novagen, Madison, Wisconsin. The latter strain served as a control group without detectable PAL / TAL activity.

As an aqueous culture pETAL and pET-24d E. Manufacturing and growing coli

In order to grow pETAL and pET-24d strains in liquid culture, 5 mL of LB medium with 25 mg / L kanamycin and 1 mM IPTG was inoculated with each strain from the glycerol stock and shaken at 300 RPM. Growing overnight at 38 ° C. The next morning, when the OD (600 nm) of the culture reached ˜2.0, the culture was centrifuged at 10,000 RPM for 10 minutes and the pellet in 4 mM phenylalanine adjusted to ˜pH 7 with water or KOH. Resuspend. The mixture was further incubated at 38 ° C. and 1 mL aliquots were taken at 6 and 24 hours. These were stored on ice before analysis by ToF-SIMS and HPLC.

Mercury Lee. Preparation of E. coli culture samples and introduction into vacuum chamber for ToF-SIMS analysis:

10 μL of an unfiltered, unmodified aqueous culture of pETAL grown for 6 and 24 hours in the presence and absence of phenylalanine was removed from a clean silicon wafer piece (Virginia Semiconductor, Fredericksburg, VA) using an Eppendorf pipette system, respectively. Moved to. The droplets were allowed to evaporate in air and then a piece of silicon wafer was placed on a stainless steel circular “puck” and introduced into a PHI model 7200 ToF-SIMS instrument (Physical Electronics, Eden Prairie, MN) as described previously.

ToF-SIMS detection of cinnamic acid product from pETAL aqueous culture:

The size of each evaporated aqueous culture microdrop on the silicon wafer was about 5 mm in diameter. The 200 x 200 μm ² area of the dried microdroplets was analyzed using a cesium main ion source, pulsed electron ejection gun for charge compensation, and an ion reflector type spectrometer. Negative ToF-SIMS data from 6-hour and 24-hour phenylalanine-supplied cultures showed molecular from secondary ions at m / z 164 showing phenylalanine (FIGS. 6B, D) and the leucine from TAL enzyme conversion of phenylalanine. Molecular secondary ions at m / z 147, representing the Namsan product. The intensity of the cinnamic acid ion peak relative to the phenylalanine ion peak was greater for the 24 hour culture compared to the 6 hour culture. None of the ions at m / z 164 or 147 were found in 6-hour and 24-hour cultures without phenylalanine (Figure 6A, D).

HPLC detection of cinnamic acid product from pETAL aqueous culture:

Aqueous cultures of pETAL and pET-24d resuspended in phenylalanine and incubated for 6 or 24 hours were subjected to HPLC analysis for cinnamic acid production. 0.1 mL of each aqueous mixture was microfuge for 10 minutes to remove cells and 10 μL of supernatant was Hewlett-Packard equipped with Zorbax ODS column (3 μm particles, 6.2 mm × 8 cm column). Hewlett-Packard) Model 1050 HPLC was injected and developed over 10 minutes with a linear gradient of acetonitrile in water containing 0.1% formic acid (5% to 80% acetonitrile in 10 minutes). Cinnamic acid was detected by uv absorption and quantified against Cinnamic acid standard (Sigma Chemical, St. Louis, MO).

The 6 hour aqueous culture of pETAL contained 0.9 mM cinnamic acid and the 24 hour sample contained 1.6 mM. No cinnamic acid was detected in the samples from the pET-24d culture. Thus, HPLC detection of cinnamic acid was associated with ToF-SIMS detection of cinnamic acid in 6 and 24 hour samples of pETAL.

PETAL and pET-24d E. coli on Supor® polyethersulfone membranes. Preparation and growth of coli colonies:

Spread 30 μL of 100 mM IPTG on a Petri dish (10 cm diameter) of LB agar containing 25 mg / L kanamycin, allow to air dry for 5 minutes and then 90 mm round Sufor® Polyether. The sulfone membrane was placed on it. Glycerol stocks of pETAL and pET-24d cultures were diluted sufficiently to ˜600 colonies per plate and pure pETAL, pure pET-24d and 1: 1 mixtures were spread over the membrane surface, respectively. After growing overnight at 38 ° C., the membranes with each type of colony on top were transferred to agar containing 4 mM phenylalanine adjusted to pH 7 with KOH and further incubated at 38 ° C. for 24 hours.

Introduction of colonies into vacuum chambers for ToF-SIMS analysis:

A 1 cm ² portion of each analytical Supor® polyethersulfone membrane was cut again, peeled off the growth medium and placed on a stainless steel circular "puck" by screwing a molybdenum mask onto the membrane. The following samples were introduced into a PHI model 7200 ToF-SIMS instrument (Physical Electronics, Eden Prairie, MN) as previously described.

Based on spectroscopic differences, ToF-SIMS distinction of pETAL versus pET-24d colonies:

Eleven individual colonies were analyzed on a membrane with a mixture of pETAL and pET-24d colonies transferred to medium containing phenylalanine using a cesium main ion source, pulsed electron ejection gun for charge compensation, and an ion reflector type spectrometer. It was. The area taken for each colony was 200 × 200 μm ² .

Secondary ions on cinnamic acid negative molecules at m / z 147 were not observed in ToF-SIMS spectra obtained from pETAL colonies grown on polyethersulfone membranes. Average-centered positive and negative ToF-SIMS data and performed on Principal Component Analysis (PCA) (PLS_Toolbox Version 2.0, Eigenvector Research, Inc., Manson, WA) associated with MATLAB Version 5.3, The Mathworks, Inc., Natick, MA. Used as input data. The output of the analysis was then fed to a multivariate curve separation routine (PLS_Toolbox, MATLAB). The treatment, familiar to those skilled in the art, has identified factors indicative of variation in spectral data. Factor 1 and Factor 2 showed different intensities, or “concentrations” for the 11 colonies analyzed (FIG. 6A). Specifically, colonies 1, 5, 8, 9, and 11 are expressed to different degrees by factor 1 but not by factor 2, while colonies 2, 3, 4, 6, 7 and 10 are assigned to factor 2 By different degrees, but not by factor 1. The negative ToF-SIMS calculated spectra associated with these two factors showed a major spectral difference in fatty acid distribution between about 150 and 350 m / z (FIG. 7B, C). The ToF-SIMS data thus identified two distinctly different colony types.

The same assay was performed on pETAL and pET-24d colonies grown on separate polyethersulfone membranes transferred to media with phenylalanine. In the collected data, pETAL colonies were represented by factor 2 and pET-24d colonies were expressed by factor 1. In addition, negative ToF-SIMS data from pETAL and pET-24d colonies showed the spectral patterns represented by factor 2 and factor 1, respectively. Thus, colonies that produce cinnamic acid and colonies that do not produce cinnamic acid were distinguished when grown on membranes in separate populations, and also when growing in populations of combined and arrayed organisms. ToF-SIMS was treated to produce low molecular weight water soluble products (cinnamic acid) without direct detection of product signals. Distinguishing between the coli was possible.

Example 4

this. ToF-SIMS detection of five β-galactosidase-cut products in E. coli

This example is based on β-galactosidase-cleaving of the reactants. Shows the utility of ToF-SIMS analysis on the range of products synthesized by E. coli. The product was methyl umbelliferone (MU) and 2-nitrophenyl-β-D prepared from the cleavage of 4-methylumbeliferyl-β-D-galactopyranoside (MUG, Sigma, ST. Louis, MO). Ortho-nitrophenol (NP) prepared from cleavage of galactopyranoside (2NPG, Sigma, St. Louis, MO), as well as 3,4-cyclohexeneoesculetin-β-D-gal Lactopyranoside (S-Gal; Sigma, St. Louis, MO), 5-bromo-3-indolyl-β-D-galactopyranoside (Bluo-Gal; Sigma, St. Louis, MO) And metabolites of X-Gal (Sigma, St. Louis, Mo.). The MU, NP and S-Gal products had similar molecular weight and solubility in cinnamic acid. E. coli expressing β-galactosidase (GAL) without the need to provide a reactant. E. coli does not express GAL. The ability to distinguish from E. coli has also been demonstrated.

E. coli on the Supor® polyethersulfone membrane for the preparation of β-galactosidase products. Preparation and growth of coli colonies

E. described in Example 1. From E. coli library, a single blue color (GAL ⁺⁾ and one of the white (GAL ^-) while taking the colonies, and inoculated into a liquid LB culture medium of 5 mL is ampicillin 100 μg / ml supplemented with shaking at 300 RPM 37 E. coli expressing β-galactosidase activity by growing overnight at & lt ; RTI ID = 0.0 &gt; E. coli (“GAL ⁺ ”) and E. coli without β-galactosidase activity. Pure cultures of E. coli (“GAL ⁻ ”) were prepared. The culture was proved to be pure GAL ⁺ or GAL ⁻ by plating on LB medium with ampicillin, IPTG and X-Gal, respectively, as in Example 1. The GAL ⁺ and GAL ⁻ cultures were spread on the Supor® polyethersulfone membrane, respectively, in contact with the growth medium. The basal growth medium consisted of ˜20 mL LB agar with 100 μg / mL ampicillin covered with 30 μL of 100 mM isopropylthiogalactoside (IPTG) aqueous solution. Each tooth. For E. coli strains, one membrane was prepared on the growth medium without any reactants and one membrane on the growth medium containing each of the five reactants. When the reaction was added, 40 μL of solution was placed on the basal medium and allowed to air dry for 15 minutes. X-gal (20 mg / mL), Bluo-Gal (20 mg / mL), MUG (10 mg / mL) and 2 NPG (50 mg / mL) were dissolved in dimethylformamide. S-Gal (20 mg / mL) was dissolved in water. The plate was incubated overnight at 38 ° C.

Introduction of colonies into vacuum chambers for ToF-SIMS analysis:

For each array a 1 cm ² portion of the Supor® polyethersulfone membrane was cut, peeled off from the growth medium and placed on a stainless steel circular "puck" by screwing the molybdenum mask onto the membrane. The following samples were introduced into a PHI Model 7200 ToF-SIMS instrument (Physical Electronics, Eden Prairie, MN) as previously described.

E. with or without MUG or NPG, with or without β-galactosidase. Coli's ToF-SIMS distinction:

this. E. coli GAL ⁺ -free of reactants. E. coli GAL ⁺ + MUG, this. E. coli GAL ^-- free of reactants, and 2. Coli GAL ^- the three or four colonies on each array of MUG +, were analyzed by the main cesium ion source, the charge compensation pulsed electron ejection gun and the ion spectrometer of the reflector type for. The area taken for each colony was 200 × 200 μm ² . Negative ToF-SIMS data was normalized and averaged and then used as a dataset for Principal Component Analysis (PCA) (PLS_Toolbox, Eigenvector Research, Inc., Manson, WA) as known to those skilled in the art.

The PCA scored each major component for each sample, indicating the importance of each major component for each sample. For each colony the scores for major components # 1, 2 and 3 were plotted on a three-dimensional map (FIG. 8). In the figure, each kind of tooth. Circles were drawn around the set of 3-4 colonies for the coli and reactant conditions. There was no overlap between the circles, meaning that the data distinguished between colony sets. ToF-SIMS distinguished:

Both E. coli expressing β-galactosidase enzyme when the MUG reactant is supplied. Coli and Toothless. Coli.

E. expressing β-galactosidase enzyme, even if no reactant was introduced. Coli and Toothless. Coli.

E. coli expressing β-galactosidase enzyme supplied with a MUG reactant. E. coli expressing β-galactosidase enzyme not supplied with the MUG reactant. Coli.

Namely, E. expressing β-galactosidase enzyme. Coli and Toothless. Distinguishing E. coli occurred in the presence and absence of the MUG reactant.

this. E. coli GAL ⁺ + NPG. Coli GAL ^- were analyzed three or four colonies on each array of + NPG in the same conditions as in the ToF-SIMS. This data reminds you of this. E. coli GAL ⁺ -free of reactants and 2. E. coli GAL ^-- Reactant free data were combined and analyzed as above. Principal Components # 1, 2 and 3 (different from Principal Components # 1, 2 and 3) for the dataset were plotted as above (FIG. 9). ToF-SIMS data distinguished:

Both E. coli expressing β-galactosidase enzyme when NPG reactants were supplied. Coli and Toothless. Coli.

E. expressing β-galactosidase enzyme, even if no reactant is introduced. Coli and Toothless. Coli.

Ie , E. coli expressing β-galactosidase . Coli and Toothless. Distinguishing E. coli occurred in the presence and absence of NPG reactants. E. coli expressing β-galactosidase enzyme fed NPG reactant. E. coli and NPG reactants expressing β-galactosidase enzymes not supplied. Circles around the sample set for E. coli overlap, meaning that ToF-SIMS did not distinguish between the colony forms. The results demonstrate that the β-galactosidase enzyme reaction product with NPG is expected to be pumped out as it is produced since it is 2-nitrophenol that is not stable in vacuum. The fact that the PCA of the ToF-SIMS data confirmed these expectations supported the value of the ToF-SIMS analysis.

this. E. coli GAL ⁺ + S-Gal and Yi. E. coli GAL ⁺ + Bluo-Gal and Lee. Two to four colonies on each array of E. coli GAL ⁺ + X-Gal were analyzed with the same ToF-SIMS conditions as above. Voice ToF-SIMS data is the same. E. coli GAL + free of reactants, E. E. coli GAL ⁺ + MUG and this. The analysis was performed as above in combination with negative ToF-SIMS data from E. coli GAL ⁺ + NPG data. Negative ToF-SIMS data was normalized and averaged and then used as input data for principal component analysis (PCA) and multivariate curve separation (MCR) (PLS_Toolbox, Eigenvector Research, Inc., Manson, WA). The treatment, which is familiar to those skilled in the art, has identified factors related to the variation in the spectral data. Factors 1, 2, 3, 4 and 5 showed different intensities or “concentrations” for the colonies analyzed (FIG. 10). Specifically, factor 1 is E. E. coli GAL ⁺ no reactants and 2. E. coli GAL ⁺ + NPG type colonies were expressed, factor 2 is E. coli. E. coli GAL ⁺ + MUG type colonies, factor 3 is E. E. coli GAL ⁺ + represents a colony of Bluo-Gal type, factor 4 is E. E. coli GAL ⁺ + S-Gal type colonies were expressed, factor 5 is E. E. coli GAL ⁺ + X-Gal type colonies were expressed. Negative ToF-SIMS calculated spectra associated with the five factors showed a major spectral difference (FIG. 10). Thus, ToF-SIMS data identified five distinctly different colony types. By factor 1. E. coli GAL ⁺ and Yi. The expression of E. coli GAL ⁺ + NPG is this. E. coli GAL ⁺ + NPG. This is consistent with the results from the aforementioned PCA alone, which showed overlap between E. coli GAL ⁺ reactant free.

The detection of secondary ions on the product negative molecular is performed by E. coli expressing β-galactosidase with the reactants Bluo-Gal, S-Gal and X-Gal. It provided a basis for the distinction of E. coli. Specifically, factor 3 showed significant properties near m / z 420, which is consistent with the molecular weight of the dimer of the cleaved product from Bluo-GAL. Factor 4 showed significant properties at m / z 231 which matched the molecular weight of the cleaved product of S-GAL. Factor 5 showed significant properties at m / z 486, consistent with the molecular weight of the dimer of the cleaved product of X-GAL.

this. The spectral pattern for the E. coli GAL + + MUG colony type was easily distinguished from other colony types, but the spectral pattern did not show molecular ions for MU. ToF-SIMS data provided specific spectral patterns related to MU production. This is similar to the case where cinnamic acid discussed in Example 3 is produced.

That is, through detection of product-specific secondary ions or through recognition of specific spectral patterns, ToF-SIMS results in a range of E. coli. It was shown that coli-synthesized products could be analyzed.

Example 5

Yarrowy lipolytica, which produces different fatty acid profiles in an array ( Yarrowia lipolytica ) ToF-SIMS detection of colonies

This example relates to a Yarrowia lipolytica strain that has been treated to produce a fatty acid profile different from that of wild type Yarrowia, which typically requires extensive sample preparation and subsequent gas chromatography analysis to distinguish. The ToF-SIMS method can distinguish between the strain and wild type based on detection of differences in fatty acid composition. This example also shows the preparation and analysis of colony copies, a technique that can be used to analyze collections of organisms by ToF-SIMS for use in analyzing mutation collections.

Yeast Strains:

The yeast strain Yarowiya lipolytica used in this example had a different fatty acid composition, each of which was a choice to allow nutritional requirements. A wild type strain called Q was obtained from ATCC under accession number 76982. It mainly accumulates oleic acid (C18: 1) and linoleic acid (C18: 2) and is a leucine nutritional supporter (phenotype: leu ⁻ ). The Q + 4 strain was derived from Q by transforming with four genes (delta 6 desaturase, elongase, delta 5 desaturase, delta 17 desaturase) to convert linoleic acid to eicosapthenic acid. It accumulates 10-15% of gamma linolenic acid (C18: 3) as well as oleic and linoleic acid and is a uracil nutrient (phenotype ura ⁻ ).

Q + 4 strain expression:

The Q + 4 strain was wild type Yarrowia lipolytica (ATCC # 76982) transformed with a pGEM-T easy vector (Promega, Madison, WI) containing a 10.3 kb DNA fragment (SEQ ID NO: 1) comprising the following sequence: )to be:

1) 440 bp 5'-non-coding DNA sequence upstream from the Yarrowia liporica URA3 gene

2) 418 bp TEF promoter of the Yarrowia lipolytica genome (from the Translation Extension Factor gene) (Muller S. et al., Yeast, 14: 1267-1283 (1998)), Mortierella alpina (Accession # AF465281) A chimeric gene comprising a 973 bp sequence containing the coding region of a high affinity PUFA elongase gene, and a 179 bp XPR2 transcription terminator (from the extracellular protease gene).

3) A chimeric gene comprising the TEF promoter, a 1357 bp sequence containing the coding region of the M. alpina Δ5 desaturase gene, and an XPR2 transcription terminator.

4) 2.25 kb sequence containing the Yarrowia LEU2 gene

5) A chimeric gene comprising a TEF promoter, Saprolegnia diclina (ATCC # 56851) Δ17 desaturase coding region, and an XPR2 transcriptional terminator codon optimized for expression in Yarrowia .

6) A chimeric gene comprising a TEF promoter, M. alpina Δ6 desaturase coding region, and an XPR2 transcription terminator.

7) 280 bp 3'-sequence of Yarrowia liporica URA3 gene.

Manufacturing / Growth of Colony Plates:

Cultures of Q and Q + 4 were grown on minimal medium prepared as follows:

Yarrowy Minimum Badge m (MM):

20 g / L glucose

Yeast nitrogen base without 1.7 g / L amino acid or ammonium sulfate

1 g / L proline

0.1 g / L Lysine

0.1 g / L adenine

The pH was adjusted to 6.1. For solid medium, 1.5% agar was added. 0.1 g / L of leucine was added for growth of the Q leucine nutritional supplement (MML); 0.1 g / L uracil and 0.1 g / L uridine were added (MMU) for growth of the Q + 4 uracil nutrient. The combined medium that allowed the growth of both Yarrowia strains contained the same concentrations of leucine, uracil and uridine (MMUL).

Glycerol stocks were used to prepare mixed cultures of Q, Q + 4, and additional strains. Mixed cultures were spread on MMUL medium and grown at 30 ° C. for 2 days. Next a 10 cm diameter plate with about 1000 colonies was replicated by placing a circular sterile Suform® 200 polyethersulfone membrane on top of the culture dish until completely wet (the filter is observed to be uniformly blacked). It was. The membrane was then removed from the dish, turned over with the side in contact with the colony on top, and then placed on a fresh dish of MMUL medium. After incubation overnight at 30 ° C., the membrane had a pattern of colonies that were exact replicas of those on the original culture plate. Additional replicate membranes were made by repeating the above procedure. Two marker points were made with a Sanford Sharpie Fine Point Blue permanent marker with lines indicating notches cut from both original and replica films. The notch corresponded to the position of the line on the edge of the original plate of the medium, which later aided in the alignment of the membrane, growth plate and ToF-SIMS data. The notch is called a radial registration label.

Introduction of colonies into vacuum chambers for ToF-SIMS analysis:

One entire Supo® polyethersulfone replica membrane was removed from the growth medium and placed on a stainless steel circular holder by fixing with a stainless steel ring that overlaps the edges of the membrane with the holder. The sample was then introduced into an ion-ToF model IV ToF-SIMS instrument (Ion-ToF, GmbH, Muenster, Germany) according to the manufacturer's instructions. After depressurizing the pump from atmospheric pressure to <5 x 10 ^-6 Torr in the pre-chamber, the sample stage was pushed into the main analytical chamber with a pressure of <5 x 10 ^-7 Torr using a magnetic transfer bar.

Use ToF-SIMS image data for colony analysis:

Colony analysis was performed using a gold main ion source, pulsed electron ejection gun for charge compensation, and an ion reflector type spectrometer (Ion-ToF Model IV, Ion-ToF, GmbH, Muenster, Germany). The protocol for analysis was described in Example 2, "Colonial Analysis with ToF-SIMS Secondary Ion Mapping Through a Stage Raster Under a Major Ion Beam." The sample stage was rastered over a 20 x 20 mm ² area with 256 x 256 pixel resolution. Full negative secondary ion mass spectra from 0-800 m / z were obtained at each approximately 78 × 78 μm ² area pixel. Then, the intensity of a particular peak from the mass spectral data at each pixel was used to create a spatial distribution map.

Since phosphate functional groups are related to the cell membrane, the nominal sum of the phosphate-related peaks at m / z 63 (PO2-) and 79 (PO3-) for each pixel maps the locations of all colonies within the analyzed area. (FIG. 10A). Overlaid phosphate maps of various colors on the map of peak intensity corresponding to the marker points were made for easy registration with the original colony plate. The spectral data can be extracted from single or multiple pixels that make up individual colonies. Negative ToF-SIMS spectral data was extracted from the series of pixels that make up the various colonies observed in the phosphate map from FIG. 11A. Three different spectral patterns were observed in the fatty acid regions of the data for the analyzed colonies (FIGS. 11A-C), two of which represented colonies of Q and Q + 4 strains (FIG. 11D). Referring to Figures 11A-D, the letters in the upper panel represent colonies from the spectrum shown in Figure 11D, and the numbers in the lower panel represent colonies taken for identification. Note that colonies labeled A are the same as labeled 1 in the middle panel, and colonies labeled B are the same as labeled 16 in the lower panel.

The third pattern represented another strain in the mixture not shown here. One spectral pattern had peaks at nominal m / z 279 and 281 consistent with the production of linoleic acid (C18: 2) and oleic acid (C18: 1) fatty acids, respectively, confirming that the colony was a Q strain. The second pattern showed a significant increase in m / z 281 intensity relative to m / z 279 intensity, and also nominally peaked at m / z 277, indicating the production of gamma linolenic acid (C18: 3), which was the colony. It was confirmed that the Q + 4 strain.

Based on the spectral differences, a map of secondary ionic strengths per pixel was generated, indicating that these colonies belonged to Q, Q + 4, and third strains. Specifically, a grayscale map of phosphate intensity / pixel was created to show the location of all colonies; A color overlay (blue) map of m / z 279 intensity / pixel with an m / z 281 intensity / pixel ratio was created to show the location of the Q colony; A color overlay (green) map of m / z 281 intensity / pixel with an m / z 279 intensity / pixel ratio was created to show the position of the Q + 4 colonies and the third spectral pattern was also overlayed (red). These maps enabled the identification of Q and Q + 4 colonies based on the color mapping of the colony arrays.

Principal component analysis as described above was performed on the standardized and mean-centered data for the dataset. The map positions and spectra of Principal Component 1 and Principal Component 2 are shown in the upper and lower panels of FIG. 12, respectively. The data distinguished different types of Yarrowia colonies.

Identification of colonies by nutritional analysis / culture on specific media

In order to trace back the colony confirmation performed as above to the original master plate of colonies on the agar surface, the size of the FIG. 11A image was reduced to 2 cm square (actual size of the replicated filter analyzed) and printed on transparent paper. The transparency image was placed underneath the original master plate of the colonies on the light box (surface facing up), and the image of the colonies visible through the transparency was visually aligned with the colonies on the plate. Colony replication scanned by ToF-SIMS was originally a mirror image of the master plate, so proper orientation was important for accurate alignment. The (mirror image) of the scan was then aligned using a radial registration mark on the plate and a marker point pointing to the mark and moved until the pattern of colonies exactly matched the image.

Once the pattern was aligned with the actual colonies, representative guidelines were taken from the master plate for subsequent confirmation of their identification using this guideline. Based on the color overlay of the error of the different spectra described above, colonies that appear mainly in blue were identified by Q, and numbers 1-12 in FIG. 11A were taken for testing. Mainly green colonies with numbers 13 to 18 in FIG. 11A were similarly taken for testing, identified as Q + 4. The method for subsequent identification of colonies taken utilized the different nutritional requirements of the strains. The Q strain needs leucine for growth and Q + 4 needs uracil. Thus, Q grows only on the MMUL and not on the MMU or MM. Q + 4 grows on the MMU but not on the MM. Colonies taken were tested by inoculation on a medium that allowed growth based on the above identification, and the cells obtained were seeded on more restrictive medium.

1. Colonies 1-12 were inoculated on solid MMUL medium. They all grew up. Next, the obtained cells were seeded on MMU and MM medium. Ten of the 12 colonies did not grow in MMU or MM, confirming their identity to be Q. Colony 6 grew in both MMU and MM, suggesting that it is not Q. Colony 11 grew to a small number of discontinuous colonies on MM and MMU, suggesting that it was slightly contaminated but nearly Q. Therefore, ToF-SIMS mapping correctly identified colonies of Q strains in 11 of 12 cases.

2. Colonies 13-18 were inoculated on solid MMU medium. They all grew up. Next, the obtained cells were seeded on MM medium. Four of the six colonies did not grow on MM, confirming that they were Q + 4. Colony 14 grew on MM, suggesting that it is not Q + 4. Colony 15 grew to a small number of discontinuous colonies on the MM, suggesting that it was slightly contaminated but nearly Q + 4. Therefore, ToF-SIMS mapping accurately identified colonies of Q + 4 strain in 5 of 6 cases.

Example 6

ToF-SIMS detection of Yarrowy Lipolytica colonies after saponification on a Supor® filter

This example shows the processing of colonies on a filter by a treatment that enhances the ability to measure ToF-SIMS detectable products. The process used to change the colonies and their products was KOH base-catalyzed hydrolysis (saponification) of the constituent lipids. When performed in solution, the treatment produces free fatty acid salts.

Preparation of saponified colonies:

Only the Q + 4 strain of yeast Yarowiya lipolytica was used in this example. Two solid medium plates of MMU medium with sterile Supor® 200 polyethersulfone membranes were inoculated from a glycerol stock of Q + 4 and grown for 48 hours at 30 ° C.

For the saponification process, one of the Supor® filters with ˜500 Yarrowia colonies was removed from the medium and air-dried for 2 minutes and then saturated with a solution of 90% methanol, 10% water and 0.3 M KOH. Filter paper blotter (blotter, Whatman No. 1 in two layers, 9 cm diameter). The filter paper was prewashed by rinsing with methanol and air drying. A Supor® filter was placed on the blotter paper for up to 1 minute and then transferred to a hybridization tube (Corning, 35 mm x 100 mm). Supor® filter was attached to the wall of the tube. The tube was tightly sealed and completely immersed in an 80 ° C. water bath. After 1 hour in the water bath, the tube was taken out and opened and allowed to air dry before removing the filter from the tube. Colonies lost most of their vertical limits and appeared as shiny yellow spots.

Introduction of colonies into vacuum chambers for ToF-SIMS analysis:

For treated arrays, and for untreated arrays, the 1 cm ² portion of the Supor® polyethersulfone membrane is cut and peeled from the growth medium to form part of the stainless steel multi-sample holder. The mask is placed upside down. The sample was then introduced into an ion-ToF model IV ToF-SIMS instrument (Ion-ToF, GmbH, Muenster, Germany) as described above.

Enhancement of ToF-SIMS negative secondary ion signal from Q + 4 Yarrowia colonies as a result of the saponification process:

Colonies were respectively analyzed using the following ToF-SIMS conditions: gold main ion source, pulsed electron ejection gun for charge compensation, and ion reflector type spectrometer. The collected colony area was 250 x 250 μm ² for unsaponified samples and 300 x 300 μm ² for saponified samples. This difference in assay area was the result of different colony sizes.

The improvement of the negative secondary ion signal in the fatty acid region was measured as follows: The area from m / z 250-290 containing C16 and C18 fatty acids was expressed as the ratio of total anion yield. An increase in this ratio of 1.7 was observed for spectra from saponified samples relative to untreated samples (FIG. 13).

In another experiment, an air dried Supor® filter with Yarrowia colonies was used to filter paper blotting paper (Wattman No. 1, 9 cm diameter) saturated with 0.3 M KOH aqueous solution without methanol. Placed on top. The filter was incubated and analyzed as described above. This treatment with KOH alone provided an improvement in signal / noise ratio similar to that obtained using KOH with methanol. Removing methanol may be beneficial because it lowers the likelihood that dissolved fatty acids will diffuse from the original point of the colony during processing.

Example 7 (expected)

Identification of Specific Microorganisms by ToF-SIMS Analysis of Fatty Acid Compositions

This example shows the identification of specific microorganisms with distinct fatty acid profiles based on ToF-SIMS analysis of fatty acid profiles for organisms.

background:

Fatty acid analysis has been used to characterize many bacteria and fungi (see, for example, Komagata, K. & Suzuki, K. (1987). Lipid and cell-wall analysis in bacterial systematics.Methods in Microbiology , 19: 161-207); Stead, DE, Sellwood, JE, Wilson, J. & Viney, I. (1992). Evaluation of a commercial microbial identification system based on fatty acid profiles for rapid, accurate identification of plant pathogenic bacteria. Journal of Applied Biochemistry , 72: 315-321.); and Welch, DF (1991). Applications of cellular fatty acid analysis. Clinical Microbiology Reviews , 4: 422-438.).

In some cases, a particular microorganism has a very characteristic fatty acid profile that diagnoses the organism's response to the presence of that organism or to specific environmental conditions. For example, the Gilardi rod group 1 bacteria commonly found in human wounds has a distinct fatty acid profile (Moss et al., Journal of Clinical Microbiology (1993) 31: 689-691). The food hospital group Listeria monocytogenes has a uniquely high composition of certain branched chain fatty acids (Moss et al., Journal of Clinical Microbiology (1993) 31: 689-691). Extraction and analysis of fatty acids by conventional techniques, such as gas chromatography of fatty acid methyl esters, is tedious and time consuming, especially when it is to be performed on a large number of samples. Using the method outlined here, much faster analysis of fatty acids of microorganisms is possible.

Microbial samples are collected by standard methods and grown into colonies on a Supor® polyethersulfone membrane filter. With the addition of the radial registration label and marker point, replication of the colony array is performed as described in Example 5. Colony-arrayed filters are placed in a ToF-SIMS instrument and analyzed as described in Example 5. Principal component analysis is performed as described above on standardized, mean-centered data. The assay is used to identify the fatty acid composition of the microorganisms in colonies on the filter. Based on the fatty acid composition of the individual colonies, the presence of certain microorganisms is identified.

                         SEQUENCE LISTING <110> E. I. DU PONT DE NEMOURS AND COMPANY   <120> DIRECT DETECTION METHOD FOR PRODUCTS OF CELLULAR METABOLISM USING        ToF-SIMS <130> CL2358 PCT <150> US 60/502151 <151> 2003-09-11 <160> 1 <170> PatentIn version 3.2 <210> 1 <211> 10328 <212> DNA <213> synthetic <400> 1 tatcacatca cgctctcatc aagaatactt cttgagaacc gtggagaccg gggttcgatt 60 ccccgtatcg gagtgtttat tttttgctca accataccct ggggtgtgtt ctgtggagca 120 ttctcacttt tggtaaacga cattgcttca agtgcagcgg aatcaaaaag tataaagtgg 180 gcagcgagta tacctgtaca gactgtaggc gataactcaa tccaattacc ccccacaaca 240 tgactggcca aactgatctc aagactttat tgaaatcagc aacaccgatt ctcaatgaag 300 gcacatactt cttctgcaac attcacttga cgcctaaagt tggtgagaaa tggaccgaca 360 agacatattc tgctatccac ggactgttgc ctgtgtcggt ggctacaata cgtgagtcag 420 aagggctgac ggtggtggtt cgtacgttgt gtggaattgt gagcggataa caatttcaca 480 caggaaacag ctatgaccat gattacgcca agctcgaaat taaccctcac taaagggaac 540 aaaagctgga gctccaccgc ggacacaata tctggtcaaa tttcagtttc gttacataaa 600 tcgttatgtc aaaggagtgt gggaggttaa gagaattatc accggcaaac tatctgttaa 660 ttgctaggta cctctagacg tccacccggg tcgcttggcg gccgaagagg ccggaatctc 720 gggccgcggt ggcggccgct tactgcaact tccttgcctt ctccttggca gcgtcggcct 780 tggcctgctt ggccaacttg gcgttctttc tgtaaaagtt gtagaagaga ccgagcatgg 840 tccacatgta gaaccaaagc agagccgtga tgaagaaggg gtatccgggg cggccaagga 900 ccttcatggc gtacatgtcc caggaagact ggaccgacat catgcagaac tgtgtcatct 960 gcgagcgcgt gatgtagaac ttgatgaacg acacctgctt gaagcccaag gccgacaaga 1020 agtagtagcc gtacatgatc acatggatga acgagttcaa cgcagcagag aagtaggctt 1080 caccgttggg tgcaacaaag gtgaccaacc accagatggt gaagatggag ctgtggtggt 1140 aaacgtgcaa gaaggagatc tggcggttgt tcttcttgag gaccatgatc atggtgtcga 1200 caaactccat gatcttggag aagtagaaga gccagatcat cttggccata ggaagaccct 1260 tgaaggtatg atcagcagcg ttctcaaaca gtccatagtt ggcctgataa gcctcgtaca 1320 ggatcccacc gcacatgtag gcgctgatcg agaccagaca aaagttgtgc aggagcgaaa 1380 acgtcttgac ctcgaaccgc tcaaagttct tcatgatctg catgcccaca aagaccgtga 1440 ccaaataagc gagcacgatc aacagcacgt ggaacgggtt catcaacggc agctcacggg 1500 ccaaaggcga ctccaccgcg accaggaacc cacgcgtgtg atggacaatc gtggggatgt 1560 acttctcggc ctgggccacc agcgcggcct cgagaggatc gacatagggc gcggcccgga 1620 caccgatagc ggtggcaagg tccataaaca gatcttgcgg catctttgat gggaggaatg 1680 gcgcaatcga ctccatgcgg ccgctctaga actagtggat cctttgaatg attcttatac 1740 tcagaaggaa atgcttaacg atttcgggtg tgagttgaca aggagagaga gaaaagaaga 1800 ggaaaggtaa ttcggggacg gtggtctttt atacccttgg ctaaagtccc aaccacaaag 1860 caaaaaaatt ttcagtagtc tattttgcgt ccggcatggg ttacccggat ggccagacaa 1920 agaaactagt acaaagtctg aacaagcgta gattccagac tgcagtaccc tacgccctta 1980 acggcaagtg tgggaaccgg gggaggtttg atatgtgggg tgaagggggc tctcgccggg 2040 gttgggcccg ctactgggtc aatttggggt caattggggc aattggggct gttttttggg 2100 acacaaatac gccgccaacc cggtctctcc tgaagcttgt gagcggataa caatttcaca 2160 caggaaacag ctatgaccat gattacgcca agctcgaaat taaccctcac taaagggaac 2220 aaaagctgga gctccaccgc ggacacaata tctggtcaaa tttcagtttc gttacataaa 2280 tcgttatgtc aaaggagtgt gggaggttaa gagaattatc accggcaaac tatctgttaa 2340 ttgctaggta cctctagacg tccacccggg tcgcttggcg gccgaagagg ccggaatctc 2400 gggccgcggt ggcggccgcc tactcttcct tgggacggag tccaagaaca cgcaagtgct 2460 ccaaatgtga agcaaatgct tgccaaaacg tatccttgac aaggtatgga accttgtact 2520 cgctgcaggt gttcttgatg atggccagaa tatcgggata atggtgctgc gacacgttgg 2580 ggaacagatg gtgcacagcc tggtagttca agctgccagt gatgctggtc cagaggtgcg 2640 aatcgtgtgc gtaatcctgc gtagtctcga cctgcatagc tgcccagtcc ttttggatga 2700 tcccgttctc gtcaggcaac ggccactgaa cttcctcaac aacgtggttc gcctggaagg 2760 tcagcgccag ccagtaagac gacaccatgt ccgcgaccgt gaacaagagc agcaccttgc 2820 ccaggggcag atactgcagg ggaacaatca ggcgatacca gacaaagaaa gccttgccgc 2880 cccagaacat cacagtgtgc catgtcgaga tgggattgac acgaatagcg tcattggtct 2940 tgacaaagta caaaatgttg atgtcctgaa tgcgcacctt gaacgccagc agtccgtaca 3000 ggaaaggaac aaacatgtgc tggttgatgt ggttgacaaa ccacttttgg ttgggcttga 3060 tacgacgaac atcgggctca gacgtcgaca cgtcgggatc tgctccagca atgttggtgt 3120 aggggtgatg gccgagcata tgttggtaca tccacaccag gtacgatgct ccgttgaaaa 3180 agtcgtgcgt ggctcccaga atcttccaga cagtggggtt gtgggtcact gaaaagtgag 3240 acgcatcatg aagagggttg agtccgactt gtgcgcacgc aaatcccatg atgattgcaa 3300 acaccacctg aagccatgtg cgttcgacaa cgaaaggcac aaagagctgc gcgtagtagg 3360 aagcgatcaa ggatccaaag ataagagcgt atcgtcccca gatctctggt ctattcttgg 3420 gatcaatgtt ccgatccgta aagtagccct cgactctcgt cttgatggtt ttgtggaaca 3480 ccgttggctc cgggaagatg ggcagctcat tcgagaccag tgtaccgaca tagtacttct 3540 tcataatggc atctgcagcc ccaaacgcgt gatacatctc aaagaccgga gtaacatctc 3600 ggccagctcc gagcaggaga gtgtccactc caccaggatg gcggctcaag aactttgtga 3660 catcgtacac cctgccgcgg atggccaaga gtaggtcgtc cttggtgtta tgggccgcca 3720 gctcttccca ggtgaaggtt tttccttggt ccgttcccat ggtgaatgat tcttatactc 3780 agaaggaaat gcttaacgat ttcgggtgtg agttgacaag gagagagaga aaagaagagg 3840 aaaggtaatt cggggacggt ggtcttttat acccttggct aaagtcccaa ccacaaagca 3900 aaaaaatttt cagtagtcta ttttgcgtcc ggcatgggtt acccggatgg ccagacaaag 3960 aaactagtac aaagtctgaa caagcgtaga ttccagactg cagtacccta cgcccttaac 4020 ggcaagtgtg ggaaccgggg gaggtttgat atgtggggtg aagggggctc tcgccggggt 4080 tgggcccgct actgggtcaa tttggggtca attggggcaa ttggggctgt tttttgggac 4140 acaaatacgc cgccaacccg gtctctcctg aattctgcag atgggctgca ggaattccgt 4200 cgtcgcctga gtcgacatca tttatttacc agttggccac aaacccttga cgatctcgta 4260 tgtcccctcc gacatactcc cggccggctg gggtacgttc gatagcgcta tcggcatcga 4320 caaggtttgg gtccctagcc gataccgcac tacctgagtc acaatcttcg gaggtttagt 4380 cttccacata gcacgggcaa aagtgcgtat atatacaaga gcgtttgcca gccacagatt 4440 ttcactccac acaccacatc acacatacaa ccacacacat ccacaatgga acccgaaact 4500 aagaagacca agactgactc caagaagatt gttcttctcg gcggcgactt ctgtggcccc 4560 gaggtgattg ccgaggccgt caaggtgctc aagtctgttg ctgaggcctc cggcaccgag 4620 tttgtgtttg aggaccgact cattggagga gctgccattg agaaggaggg cgagcccatc 4680 accgacgcta ctctcgacat ctgccgaaag gctgactcta ttatgctcgg tgctgtcgga 4740 ggcgctgcca acaccgtatg gaccactccc gacggacgaa ccgacgtgcg acccgagcag 4800 ggtctcctca agctgcgaaa ggacctgaac ctgtacgcca acctgcgacc ctgccagctg 4860 ctgtcgccca agctcgccga tctctccccc atccgaaacg ttgagggcac cgacttcatc 4920 attgtccgag agctcgtcgg aggtatctac tttggagagc gaaaggagga tgacggatct 4980 ggcgtcgctt ccgacaccga gacctactcc gttcctgagg ttgagcgaat tgcccgaatg 5040 gccgccttcc tggcccttca gcacaacccc cctcttcccg tgtggtctct tgacaaggcc 5100 aacgtgctgg cctcctctcg actttggcga aagactgtca ctcgagtcct caaggacgaa 5160 ttcccccagc tcgagctcaa ccaccagctg atcgactcgg ccgccatgat cctcatcaag 5220 cagccctcca agatgaatgg tatcatcatc accaccaaca tgtttggcga tatcatctcc 5280 gacgaggcct ccgtcatccc cggttctctg ggtctgctgc cctccgcctc tctggcttct 5340 ctgcccgaca ccaacgaggc gttcggtctg tacgagccct gtcacggatc tgcccccgat 5400 ctcggcaagc agaaggtcaa ccccattgcc accattctgt ctgccgccat gatgctcaag 5460 ttctctctta acatgaagcc cgccggtgac gctgttgagg ctgccgtcaa ggagtccgtc 5520 gaggctggta tcactaccgc cgatatcgga ggctcttcct ccacctccga ggtcggagac 5580 ttgttgccaa caaggtcaag gagctgctca agaaggagta agtcgtttct acgacgcatt 5640 gatggaagga gcaaactgac gcgcctgcgg gttggtctac cggcagggtc cgctagtgta 5700 taagactcta taaaaagggc cctgccctgc taatgaaatg atgatttata atttaccggt 5760 gtagcaacct tgactagaag aagcagattg ggtgtgtttg tagtggagga cagtggtacg 5820 ttttggaaac agtcttcttg aaagtgtctt gtctacagta tattcactca taacctcaat 5880 agccaagggt gtagtcggtt tattaaagga agggagttgt ggctgatgtg gatagatatc 5940 tttaagctgg cgactgcacc caacgagtgt ggtggtagct tgttactgta tattcggtaa 6000 gatatatttt gtggggtttt agtggtgttt aaacgacgga attcctgcag cccatctgca 6060 gaattcagga gagaccgggt tggcggcgta tttgtgtccc aaaaaacagc cccaattgcc 6120 ccaattgacc ccaaattgac ccagtagcgg gcccaacccc ggcgagagcc cccttcaccc 6180 cacatatcaa acctcccccg gttcccacac ttgccgttaa gggcgtaggg tactgcagtc 6240 tggaatctac gcttgttcag actttgtact agtttctttg tctggccatc cgggtaaccc 6300 atgccggacg caaaatagac tactgaaaat ttttttgctt tgtggttggg actttagcca 6360 agggtataaa agaccaccgt ccccgaatta cctttcctct tcttttctct ctctccttgt 6420 caactcacac ccgaaatcgt taagcatttc cttctgagta taagaatcat tcaccatggc 6480 tgaggataag accaaggtcg agttccctac cctgactgag ctgaagcact ctatccctaa 6540 cgcttgcttt gagtccaacc tcggactctc gctctactac actgcccgag cgatcttcaa 6600 cgcatctgcc tctgctgctc tgctctacgc tgcccgatct actcccttca ttgccgataa 6660 cgttctgctc cacgctctgg tttgcgccac ctacatctac gtgcagggtg tcatcttctg 6720 gggtttcttt accgtcggtc acgactgtgg tcactctgcc ttctcccgat accactccgt 6780 caacttcatc attggctgca tcatgcactc tgccattctg actcccttcg agtcctggcg 6840 agtgacccac cgacaccatc acaagaacac tggcaacatt gataaggacg agatcttcta 6900 ccctcatcgg tccgtcaagg acctccagga cgtgcgacaa tgggtctaca ccctcggagg 6960 tgcttggttt gtctacctga aggtcggata tgctcctcga accatgtccc actttgaccc 7020 ctgggaccct ctcctgcttc gacgagcctc cgctgtcatc gtgtccctcg gagtctgggc 7080 tgccttcttc gctgcctacg cctacctcac atactcgctc ggctttgccg tcatgggcct 7140 ctactactat gctcctctct ttgtctttgc ttcgttcctc gtcattacta ccttcttgca 7200 tcacaacgac gaagctactc cctggtacgg tgactcggag tggacctacg tcaagggcaa 7260 cctgagctcc gtcgaccgat cgtacggagc tttcgtggac aacctgtctc accacattgg 7320 cacccaccag gtccatcact tgttccctat cattccccac tacaagctca acgaagccac 7380 caagcacttt gctgccgctt accctcacct cgtgagacgt aacgacgagc ccatcattac 7440 tgccttcttc aagaccgctc acctctttgt caactacgga gctgtgcccg agactgctca 7500 gattttcacc ctcaaagagt ctgccgctgc agccaaggcc aagagcgacc accaccatca 7560 ccaccattaa gcggccgcca ccgcggcccg agattccggc ctcttcggcc gccaagcgac 7620 ccgggtggac gtctagaggt acctagcaat taacagatag tttgccggtg ataattctct 7680 taacctccca cactcctttg acataacgat ttatgtaacg aaactgaaat ttgaccagat 7740 attgtgtccg cggtggagct ccagcttttg ttccctttag tgagggttaa tttcgagctt 7800 ggcgtaatcg atgcagaatt caggagagac cgggttggcg gcgtatttgt gtcccaaaaa 7860 acagccccaa ttgccccaat tgaccccaaa ttgacccagt agcgggccca accccggcga 7920 gagccccctt caccccacat atcaaacctc ccccggttcc cacacttgcc gttaagggcg 7980 tagggtactg cagtctggaa tctacgcttg ttcagacttt gtactagttt ctttgtctgg 8040 ccatccgggt aacccatgcc ggacgcaaaa tagactactg aaaatttttt tgctttgtgg 8100 ttgggacttt agccaagggt ataaaagacc accgtccccg aattaccttt cctcttcttt 8160 tctctctctc cttgtcaact cacacccgaa atcgttaagc atttccttct gagtataaga 8220 atcattcacc atggctgctg ctcccagtgt gaggacgttt actcgggccg aggttttgaa 8280 tgccgaggct ctgaatgagg gcaagaagga tgccgaggca cccttcttga tgatcatcga 8340 caacaaggtg tacgatgtcc gcgagttcgt ccctgatcat cccggtggaa gtgtgattct 8400 cacgcacgtt ggcaaggacg gcactgacgt ctttgacact tttcaccccg aggctgcttg 8460 ggagactctt gccaactttt acgttggtga tattgacgag agcgaccgcg atatcaagaa 8520 tgatgacttt gcggccgagg tccgcaagct gcgtaccttg ttccagtctc ttggttacta 8580 cgattcttcc aaggcatact acgccttcaa ggtctcgttc aacctctgca tctggggttt 8640 gtcgacggtc attgtggcca agtggggcca gacctcgacc ctcgccaacg tgctctcggc 8700 tgcgcttttg ggtctgttct ggcagcagtg cggatggttg gctcacgact ttttgcatca 8760 ccaggtcttc caggaccgtt tctggggtga tcttttcggc gccttcttgg gaggtgtctg 8820 ccagggcttc tcgtcctcgt ggtggaagga caagcacaac actcaccacg ccgcccccaa 8880 cgtccacggc gaggatcccg acattgacac ccaccctctg ttgacctgga gtgagcatgc 8940 gttggagatg ttctcggatg tcccagatga ggagctgacc cgcatgtggt cgcgtttcat 9000 ggtcctgaac cagacctggt tttacttccc cattctctcg tttgcccgtc tctcctggtg 9060 cctccagtcc attctctttg tgctgcctaa cggtcaggcc cacaagccct cgggcgcgcg 9120 tgtgcccatc tcgttggtcg agcagctgtc gcttgcgatg cactggacct ggtacctcgc 9180 caccatgttc ctgttcatca aggatcccgt caacatgctg gtgtactttt tggtgtcgca 9240 ggcggtgtgc ggaaacttgt tggccatcgt gttctcgctc aaccacaacg gtatgcctgt 9300 gatctcgaag gaggaggcgg tcgatatgga tttcttcacg aagcagatca tcacgggtcg 9360 tgatgtccac ccgggtctat ttgccaactg gttcacgggt ggattgaact atcagatcga 9420 gcaccacttg ttcccttcga tgcctcgcca caacttttca aagatccagc ctgctgtcga 9480 gaccctgtgc aaaaagtaca atgtccgata ccacaccacc ggtatgatcg agggaactgc 9540 agaggtcttt agccgtctga acgaggtctc caaggctacc tccaagatgg gtaaggcgca 9600 gtaagcggcc gccaccgcgg cccgagattc cggcctcttc ggccgccaag cgacccgggt 9660 ggacgtctag aggtacctag caattaacag atagtttgcc ggtgataatt ctcttaacct 9720 cccacactcc tttgacataa cgatttatgt aacgaaactg aaatttgacc agatattgtg 9780 tccgcggtgg agctccagct tttgttccct ttagtgaggg ttaattaatt cgatatcata 9840 attgtcggcc gaggtctgta cggccagaac cgagatccta ttgaggaggc caagcgatac 9900 cagaaggctg gctgggaggc ttaccagaag attaactgtt agaggttaga ctatggatat 9960 gtcatttaac tgtgtatata gagagcgtgc aagtatggag cgcttgttca gcttgtatga 10020 tggtcagacg acctgtctga tcgagtatgt atgatactgc acaacctgtg tatccgcatg 10080 atctgtccaa tggggcatgt tgttgtgttt ctcgatacgg agatgctggg tacaagtagc 10140 taatacgatt gaactactta tacttatatg aggcttgaag aaagctgact tgtgtatgac 10200 ttattctcaa ctacatcccc agtcacaata ccaccactgc actaccacta caccaaaacc 10260 atgatcaaac cacccatgga cttcctggag gcagaagaac ttgttatgga aaagctcaag 10320 agagagaa 10328  

Claims (23)

a) comprising a colony of biological organisms producing a product detectable by ToF-SIMS on a vacuum compatible support; b) performing ToF-SIMS analysis on the colonies of (a) to generate data; c) correlating the data of (b) with the colony of (a) to identify a biological organism that produces a ToF-SIMS detectable product. a) a mixed population of biological organisms present in a two-dimensional array on a vacuum compatible support, wherein at least one organism produces a product detectable by ToF-SIMS; b) performing ToF-SIMS analysis on the organism array of (a) to generate data; c) mapping the array to give each organism a unique locus on the array; d) identifying one or more loci on the array in which the ToF-SIMS detectable product is present; e) associating said data with the unique organism locus of (c) to identify organisms that produce ToF-SIMS detectable products. a) a mixed population of biological organisms present in a two-dimensional array on a vacuum compatible support, wherein at least one organism produces a primary product; b) contacting the array of (a) with a predetermined material under conditions such that the primary product reacts to produce a ToF-SIMS detectable product; c) performing ToF-SIMS analysis on the organism array of (a) to generate data; d) mapping the array to give each organism a unique locus on the array; e) identifying one or more loci on the array in which the ToF-SIMS detectable product is present; f) associating said data with the unique organism locus of (d) to identify an organism that produces a ToF-SIMS detectable product. a) having a mixed population of biological organisms present in a two-dimensional array on a vacuum compatible support; b) transferring the array to a secondary growth medium such that at least one incubated biological organism on the secondary growth medium produces a product detectable by ToF-SIMS; c) performing ToF-SIMS analysis on the organism array of (a) to generate ToF-SIMS data; d) mapping the array to give each organism a unique locus on the array; e) identifying one or more loci on the array in which the ToF-SIMS detectable product is present; f) associating said data with the unique organism locus of (d) to identify an organism that produces a ToF-SIMS detectable product. The method of claim 2, wherein the mapping of the array is performed with ToF-SIMS data. The method of claim 2, wherein identifying at least one locus on the array is performed with ToF-SIMS data. 5. The method of claim 2, wherein the mixed population of organisms is incubated in a two dimensional array on primary medium before being placed on a vacuum compatible support. The method of claim 1, wherein the biological organism is selected from the group consisting of prokaryotes and eukaryotes. The method of claim 8, wherein said biological organism is a plant cell. 10. The method of claim 9, wherein said biological organism is selected from the group consisting of corn, rice, wheat, soybeans, tobacco, and arabidopsis. The method of claim 8, wherein said biological organism is selected from the group consisting of yeast and bacteria. The method of claim 11, wherein the biological organism, the MY process as Saccharomyces (Saccharomyces), Pichia (Pichia), Yarrow baby.- (Yarrowia), Rhodococcus (Rhodococcus), Streptomyces (Streptomyces), evil Martino fine test (Actinomycetes), Corynebacterium (Corynebacterium), Bacillus (Bacillus), Escherichia (Escherichia), Pseudomonas (Pseudomonas), Salmonella (Salmonella), El Winiah (Erwinia), Penny room Solarium (Penicillium), Fusarium (Fusarium), Aspergillus way across to Fernand's (Aspergillus), Castello La grapes (Podospora), Creative source Four Leeum (Chrysosporium), Trichoderma (Trichoderma) and Neuro Castello La is selected from the group consisting of (Neurospora). The method of claim 1, wherein the organism forms colonies. The method of claim 1, wherein the vacuum compatible substrate is selected from the group consisting of nylon, nitrocellulose, polyethersulfone, polysulfone, polycarbonate, polystyrene, silicon / silica and glassy carbon. The method of claim 1, wherein the ToF-SIMS detectable product is selected from the group consisting of fatty acids, p-hydroxycinnamic acid, cinnamic acid, beta-galactosidase product. The method of claim 15, wherein the fatty acid is lauric acid, myristic acid, palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid, di-homo-gamma linoleic acid, arachidonic acid, stearic acid, eicosateenoic acid, Eicosapentaenoic acid, docosahexaenoic acid, hydroxy fatty acids, peroxy fatty acids, branched chain fatty acids, phospholipids and phospholipid fragments, triglycerides and triglyceride fragments. 4. The process of claim 3 wherein the primary product is released to react with the reactants by a process of saponification. 18. The method of claim 17, wherein the saponification is carried out in the presence of potassium hydroxide and in the absence of methanol. 5. The method of claim 2, wherein the unique organism locus is provided by generating an optical picture of an array that assigns a unique locus to each organism on the array. 6. 5. The method of claim 1, wherein the ToF-SIMS product is 8 kD or less. 6. The method of claim 2, wherein ToF-SIMS analysis collects data at each pixel of the two-dimensional pixel array over dimensions of the array. 5. The method of claim 1, wherein the ToF-SIMS data is refined by a chemometric method. The method of claim 22, wherein the chemistry method is selected from the group consisting of principal component analysis (PCA) and multivariate curve separation (MCR).

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